The Fanconi Anaemia Protein D2 has an Essential Role in Telomere Maintenance in Cells that Utilize the Alternative Lengthening of Telomeres Pathway

by

Heather Ann Root

A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy

Graduate Department of Molecular Genetics University of Toronto

© Copyright by Heather Ann Root 2010

ii

The Fanconi Anaemia Protein D2 has an Essential Role in Telomere

Maintenance in Cells that Utilize the

Alternative Lengthening of Telomeres Pathway

Heather Ann Root

Doctorate of Philosophy

Molecular Genetics University of Toronto

2010

Abstract

Fanconi anaemia (FA) is an inherited disorder characterized by bone marrow failure, cancer

predisposition and congenital abnormalities. The 12 known FA genes have been implicated in

homologous recombination (HR), a process involved in telomere maintenance. A complex of at

least 7 FA proteins promotes FANCD2 monoubiquitination and nuclear foci formation.

FANCD2 colocalizes and interacts with HR proteins, however the role of FANCD2 in HR is

unclear.

Telomeres in dividing human somatic cells shorten until they reach a critical length, triggering

most cells to undergo senescence or apoptosis. Rare immortal cells escape this crisis by

expressing telomerase, or activating the Alternative Lengthening of Telomeres (ALT) pathway,

which involves HR.

FA core complex proteins and FANCD2 colocalize with telomeric foci in ALT, but not

telomerase positive cells. Localization of FANCD2 to ALT telomeric foci requires

monoubiquitination by the FA core complex, but is independent of ATM and ATR.

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FANCD2 primarily colocalizes with ALT telomeric DNA within ALT-associated PML bodies

(APBs). Electron spectroscopic imaging and FISH experiments show that APBs contain extra-

chromosomal telomeric repeat (ECTR) DNA that is non-nucleosomal. Depletion of FANCD2

causes marked increases in ECTR in ALT, but not telomerase positive cells. Overexpression of

BLM, the helicase mutated in Bloom syndrome, also causes an ALT-specific increase in ECTR

DNA. FANCD2 coimmunoprecipitates with BLM in ALT cells, and FANCD2 localization to

ALT telomeric foci requires BLM expression.

FANCD2-depleted ALT cells have reduced viability, signs of mitotic catastrophe, and multiple

types of telomeric abnormalities, including increases in telomeric recombination, entanglements,

colocalization with DNA repair proteins, and expression of fragile site characteristics. SiRNA

depletion of FANCD2 does not cause overexpression of BLM, however codepletion of BLM

with FANCD2 suppresses the telomere phenotypes caused by FANCD2 knockdown. Together

this suggests that FANCD2 regulates BLM-dependent recombination and amplification of

telomeric DNA within ALT cells.

iv

Acknowledgments

I would like to thank my supervisor Dr. Stephen Meyn for allowing me to develop and explore

my ideas, providing guidance and insight when required, and providing support when it was

needed most. I would also like to thank members of the Meyn lab, past and present, for their

technical assistance during the initial stages of the project, and useful discussions during the later

periods of the project. In particular, I would like to thank Dr. Paul Bradshaw and Magan Trottier

for listening when I was excited or frustrated, and taking the time to talk about my results. In

addition, I would like to thank my family and friends for their support. A special thank you to my

husband Jamie for providing encouragement, motivation, and advice, and always finding ways to

support my hopes and dreams. A final thank you to Aidan, for providing me with the motivation

to get things done.

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Table of Contents

Table of Contents 

Acknowledgments ................................................................................................................. iv 

Table of Contents.................................................................................................................... v 

List of Figures....................................................................................................................... viii 

List of Abbreviations .............................................................................................................. xi 

Chapter 1 ..............................................................................................................................1 

1  Introduction......................................................................................................................1 

1.1  Telomere Biology ...........................................................................................................................................................1 1.1.1  Telomere Structure and Function.......................................................................................................................1 1.1.2  Telomere Maintenance Mechanisms .................................................................................................................6 1.1.3  Telomeres and Disease ......................................................................................................................................... 15 

1.2  Fanconi Anaemia (FA)...............................................................................................................................................17 1.2.1  The FA Clinical Phenotype................................................................................................................................... 17 1.2.2  FA and Telomere Maintenance ......................................................................................................................... 19 1.2.3  The FA Pathway....................................................................................................................................................... 20 1.2.4  The Role of FANCD2 in DNA Repair ................................................................................................................ 24 

1.3  Concluding Remarks..................................................................................................................................................29 1.4  References......................................................................................................................................................................31 

Chapter 2 ............................................................................................................................ 49 

2  The Fanconi Anaemia Pathway Plays a Critical Role in Cells that Utilize the Alternerative 

Pathway of Telomere Maintenance....................................................................................... 49 

2.1  Abstract ...........................................................................................................................................................................49 2.2  Introduction ..................................................................................................................................................................50 2.3  Materials and Methods .............................................................................................................................................52 2.4  Results..............................................................................................................................................................................56 2.4.1  FANCD2 localize to telomeric foci and PML bodies in ALT human cells......................................... 56 

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2.4.2  FA core complex components localize to ALT telomeric foci and promote FANCD2 

monoubiquitination and localization to telomeric foci .......................................................................................... 59 2.4.3  FANCD2 localizes to ALT telomeric foci that have not activated a DNA damage response, 

and localization to telomeric foci is independent of ATM and ATR kinase activity ................................... 62 2.4.4  FANCD2 coimmunoprecipitates with TRF2 and BLM in ALT cells, and almost always 

localizes to telomeric foci that also contain BLM...................................................................................................... 65 2.4.5  FANCD2 localization to APBs is independent of TRF2, but requires BLM expression .............. 66 2.4.6  FANCD2 knockdown causes an ALT­specific increase in telomere dysfunction induced foci 

that is independent of rapid telomere shortening .................................................................................................... 70 2.4.7  ALT­associated PML bodies (APBs) are structurally different from non­ALT bodies, and 

contain telomeric nucleic acid in the interior of the body that differs from surrounding chromatin73 2.4.8  FANCD2 depletion in ALT cells results in nuclear abnormalities, centrosome amplification 

and rapid cell death................................................................................................................................................................ 77 2.5  Discussion.......................................................................................................................................................................83 2.6  References......................................................................................................................................................................91 

Chapter 3 .......................................................................................................................... 101 

3  Fanconi Anaemia Protein D2 Limits BLM‐Dependent, RAD51‐Independent Telomeric 

Recombination and DNA Synthesis in ALT‐Immortalized Human Cells ................................. 101 

3.1  Abstract ........................................................................................................................................................................ 101 3.2  Introduction ............................................................................................................................................................... 102 3.3  Materials and Methods .......................................................................................................................................... 106 3.4  Results........................................................................................................................................................................... 109 3.4.1  FANCD2­depletion results in a rapid, ALT­specific increase in telomeric DNA synthesis .....109 3.4.2  FANCD2­depleted ALT cells accumulate ECTR DNA within and outside of abnormally large 

ALT associated PML bodies ...............................................................................................................................................112 3.4.3  FANCD2­depleted ALT cells do not upregulate expression of BLM, TRF1 or TRF2..................118 3.4.4  FANCD2­depleted ALT cells have increased association of RAD51 with telomeric foci, 

telomere sister chromatid exchanges, fragile telomeres, and telomere entanglements........................121 3.4.5  Telomere abnormalities in FANCD2­depleted ALT cells are generated through a BLM 

dependent, largely RAD51 independent mechanism .............................................................................................128 3.4.6  Codepletion of BLM with FANCD2 improves the viability of FANCD2 depleted ALT cells ....132 

3.5  Discussion.................................................................................................................................................................... 134 3.6  References................................................................................................................................................................... 144 

vii

Chapter 4 .......................................................................................................................... 154 

4  Summary and Future Directions .................................................................................... 154 

4.1  Summary and Future Directions ....................................................................................................................... 154 4.2  References................................................................................................................................................................... 166 

viii

List of Figures

CHAPTER 1

Figure 1-1. Overview of telomere binding complexes and states that telomeres may exist in....... 2 

Figure 1-2. Model of the telomere length hypothesis of cellular senescence and immortalization.7 

Figure 1-3. Potential mechanisms of extra-chromosomal telomeric repeat (ECTR) DNA

production in ALT ........................................................................................................................ 10 

Figure 1-4. Potential mechanisms of recombination based telomere elongation in ALT cells .... 14 

Figure 1-5. Overview of FA pathway conservation in eukaryotes ............................................... 22 

Figure 1-6. Model of FA pathway activation................................................................................ 24 

CHAPTER 2

Figure 2-1. FANCD2 frequently colocalizes with telomeric DNA and telomere binding proteins

in cells that utilize the ALT pathway, but not in telomerase positive cells .................................. 57 

Figure 2-2. FANCD2 primarily colocalizes with telomeric proteins within ALT-associated PML

Bodies (APBs) .............................................................................................................................. 58 

Figure 2-3 FA core complex proteins FANCA and FANCG colocalize with FANCD2 at ALT

telomeric foci ................................................................................................................................ 60 

Figure 2-4. FANCD2 localization to ALT telomeric foci in GM847 and VA13 ALT cells is

dependent on monoubiquitination by the FA core complex......................................................... 62 

Figure 2-5. FANCD2 localization to ALT telomeric foci is not simply part of a DNA damage

response......................................................................................................................................... 65 

Figure 2-6. FANCD2 interacts with BLM and TRF2 in late S/G2 ALT, but not telomerase

positive cells.................................................................................................................................. 67 

Figure 2-7 FANCD2 localization to ALT telomeric foci is independent of TRF2 expression .... 68 

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Figure 2-8 FANCD2 localization to ALT telomeric foci is dependent on BLM expression ....... 69 

Figure 2-9. FANCD2 siRNA significantly reduces FANCD2 expression ................................... 70 

Figure 2-10. FANCD2 knockdown results in an ALT-specific increase in telomere dysfunction

induced foci that is independent of telomere rapid deletion events.............................................. 72 

Figure 2-11. APBs contain nucleic acid within the body, differing from non-ALT associated

PML bodies, which are solid protein structures............................................................................ 75 

Figure 2-12. APBs in FANCD2 depleted cells tend to be physically larger then APBs in controls,

and can contain blocks of chromatin-like nucleic acid................................................................. 76 

Figure 2-13. FANCD2 depletion results in severe nuclear abnormalities in GM847 and VA13

ALT cells ...................................................................................................................................... 78 

Figure 2-14. FANCD2 depletion increases the frequency of abnormal nuclei and cells with

supernumerary centrosomes in GM847 and VA13 ALT cells, but not in GM639 and HT1080

telomerase positive cells ............................................................................................................... 80 

Figure 2-15. FANCD2 depletion leads to a decrease in cell growth and survival in ALT cells .. 81 

CHAPTER 3

Figure 3-1. FANCD2 siRNA significantly reduces FANCD2 expression ................................. 109 

Figure 3-2 FANCD2-depleted GM847 and U2OS ALT cells have increased levels of telomeric

DNA............................................................................................................................................ 110 

Figure 3-3. FANCD2-depleted GM637 and VA13 ALT cells, but not GM639 or HT1080

telomerase positive cells, have increased levels of telomeric DNA ........................................... 111 

Figure 3-4. FANCD2-depleted ALT cells accumulate high amounts of telomeric DNA and

telomere binding proteins within APBs...................................................................................... 114 

Figure 3-5. APBs in FANCD2-depleted cells contain high levels of DNA, not RNA............... 116 

Figure 3-6. Large APBs contain high amounts of single-strand DNA ....................................... 117 

x

Figure 3-7 FANCD2-depleted ALT cells accumulate ECTR DNA outside of APBs................ 119 

Figure 3-8. FANCD2-depleted ALT cells do not increase expression of BLM,

TRF1, or TRF2............................................................................................................................ 120 

Figure 3-9. FANCD2 depletion effects RAD51 foci formation and oligomerization ................ 122 

Figure 3-10. SiRNA depletion of FANCD2 in GM847 ALT cells results in increased

frequency of T-SCEs................................................................................................................... 124 

Figure 3-11. ALT telomeres more frequently exhibit FISH staining patterns characteristic of

fragile sites then telomerase positive cells.................................................................................. 126 

Figure 3-12. FANCD2-depleted GM847 ALT cells frequently show evidence of telomeric DNA

entanglements ............................................................................................................................. 127 

Figure 3-13. BLM, but not RAD51, is required to generate high levels of ECTR DNA in

FANCD2-depleted ALT cells ..................................................................................................... 129 

Figure 3-14. Codepletion of BLM with FANCD2, but not RAD51, suppresses the increase in

T-SCEs observed in FANCD2-depleted GM847 cells ............................................................... 130 

Figure 3-15. Codepletion of BLM with FANCD2, but not RAD51, suppresses the increase in

telomeric DNA entanglements observed in FANCD2-depleted GM847 ALT cells .................. 131 

Figure 3-16. Codepletion of BLM with FANCD2 partially restores the colony forming ability of

ALT cells .................................................................................................................................... 133 

xi

List of Abbreviations

ALT alternative lengthening of telomeres

APBs ALT-associated PML bodies

BrdU bromodeoxyuridine

CO-FISH chromosome orientation fluorescent in situ hybridization

DC dyskeratosis congenita

dsDNA double-strand DNA

ECTR extra-chromosomal telomeric repeat

ESI electron spectroscopic imaging

FA Fanconi anaemia

HR homologous recombination

SCE sister chromatid exchange

ssDNA single-strand DNA

TERC telomerase RNA component

TERRA telomere repeat containing RNA

TERT telomerase reverse transcriptase

TIFs telomere dysfunction induced foci

T-SCE telomere sister chromatid exchange

1

Chapter 1

1 Introduction

1.1 Telomere Biology

1.1.1 Telomere Structure and Function

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes, facilitating

replication of the ends, and preventing ends from activating cell cycle checkpoints, or becoming

involved in DNA repair pathways that could result in chromosome fusions or rapid shortening

events. In most eukaryotes, telomeres are composed of tandem repeats of a short sequence

containing repeated guanosines. In humans, this repeat sequence is TTAGGG, and it is repeated

thousands of times to yield telomeres that typically span 10-15kb at birth (de Lange et al, 1990).

Telomeres displaying strand asymmetry, with the 3’ end always enriched for guanosine and the

5’ end always enriched for cytosine, and are therefore referred to as the G-strand and C-strand,

respectively. The G-strand typically protrudes 50-300nt past the C-strand in human telomeres,

creating a 3’ single-strand DNA (ssDNA) overhang (Makarov et al, 1997; McElligott and

Wellinger, 1997). The presence and size of 3’ overhangs on both the leading and lagging strand

telomere, suggests that overhangs are not merely a byproduct of the end replication problem, but

rather are generated through active nucleolytic processing.

The 3’ overhang appears to play important roles in telomere function, and telomeres that have

lost their G-strand overhang can become fused together in a nonhomologous end-joining process

(van Steensel et al, 1998). Telomeres can be arranged in higher order structures visible by

electron microscopy, termed telomere loops (t-loops), which are formed when the 3’ssDNA

overhang invades proximal double-strand DNA (dsDNA) in cis, forming a large duplex loop

with a partial or full Holliday junction at its base (Figure 1-1 B). The size of the t-loop appears to

vary relative to telomere length, and can encompass up to 30kb of telomeric DNA in mice, but as

little as 0.3kb of telomeric DNA in trypanosomes (Griffith et al, 1999; Muñoz-Jordán et al,

2001). Formation of a t-loop is one potential way of preventing the linear end of the chromosome

from being detected as a double-strand break and activating a DNA damage response, and

2

t-loops have been detected in telomeres from mammal, plant, avian and protist species (Griffith

et al, 1999; Cesare et al, 2003; Muñoz-Jordán et al, 2001; Murti and Prescott, 1999).

A)

B)

Figure 1-1. Overview of telomere binding complexes and states that telomeres may exist in. A) Model of the shelterin telomere binding complex (left) and the CST protein complex (right). TRF1 and

TRF2 bind double-strand telomeric DNA, while POT1 binds to single-strand G rich telomeric DNA. The

CST complex is an RPA-like complex that binds to single-strand G rich telomeric DNA. B) Telomeres

may exist in linear (upper) or looped (lower) states.

In addition to t-loops, other forms of higher order structures may also play a role in telomere

capping. Under physiological conditions, 4 guanine bases can be arranged with a four-fold

rotation of symmetry, forming a G-tetrad, which can subsequently be stacked into higher order

four stranded helical structure referred to as G-quadruplexes. Formation of higher order G-

quadruplex structures on the 3’ overhang has also been proposed to act to as a protective capping

structure (Xu et al, 2009). However, both t-loops and G-quadruplex structures are likely to be

dynamic structures, as t-loops must be removed during telomere replication, and stabilization of

3

G-quadruplexes with small molecules leads to telomere dysfunction (Gomez et al, 2006).

Supporting the idea that telomere conformation is dynamic, only a fraction of isolated linear

telomeric DNA molecules can be seen forming t-loops, and during the G2 phase of the cell cycle

telomeres transiently adopt a more open structure that is accessible to addition of nucleotides by

terminal transferase (Verdun et al, 2005).

Telomere protection is also dependent on proteins that bind directly to telomeric DNA. Telomere

Repeat Binding Factors 1 and 2 (TRF1, TRF2) bind to double-strand telomeric DNA, while Pot1

binds to single-strand G rich telomeric DNA (Chong et al, 1995; Broccoli et al, 1997; Baumann

and Cech, 2001). These three DNA binding proteins associate with 3 additional proteins, TIN2,

TPP1, and RAP1, which form a 6 protein complex termed the shelterin complex (Figure 1-1 A)

(Kim et al, 1999; Liu et al, 2004; Ye et al, 2004; Li et al, 2000). Components of the shelterin

complex can aid in the capping of telomeres in multiple ways. TRF2 can stimulate formation of

t-loop structures in vitro, and also can bind directly to ATM, a key phosphatidylinositol 3-kinase

related protein that activates the DNA damage pathway in response to double-strand breaks

(Griffith et al, 1999; Karlseder et al, 2004). The region where TRF2 binds to ATM spans serine

1981, whose autophosphorylation plays an essential role in ATM activation. Overexpression of

TRF2 results in a blunting of the ATM dependent DNA damage response at telomeres, as well as

other genomic sites of introduced double-strand breaks (Karlseder et al, 2004; Bradshaw et al,

2005; Bradshaw and Meyn, upubl.; Cesare et al, 2009). Given the high local concentration of

TRF2 at telomeres, TRF2 may aid in telomere capping through direct suppression of ATM

activation.

When a cell is depleted of TRF2, telomeres are handled in a manner similar to that of double-

strand breaks. The histone H2AX variant becomes phosphorylated at serine 139, and proteins

implicated in the response to double-strand breaks accumulate at the telomere, including ATM

phosphorylated at serine 1981, the MRE11/RAD51/NBS1 complex, and the chromatin binding

factors 53BP1 and MDC1, which are core components of a megabase platform of DNA damage

response factors that assembles around the double-strand break (Takai et al, 2003; Dimitrova and

de Lange, 2006). These factors accumulate at levels detectable by immunofluorescence, forming

foci that are referred to as Telomere Dysfunction Induced Foci (TIFs). TRF2 depletion also

results in loss of the G-Strand overhang and chromosome end fusions that retain telomeric DNA

at the point of fusion, demonstrating that telomeres can become uncapped in a length

4

independent manner (van Steensel et al, 1998; Dimitrova and de Lange, 2006). Widespread

telomere dysfunction in TRF2 depleted cells is quickly followed by cell cycle arrest and

induction of apoptosis or senescence (van Steensel et al, 1998; Karlseder et al, 1999).

ATR is a phosphatidylinositol 3-kinase related protein that plays a key role in coordinating the

response to stalled and collapsed replication forks. POT1 binds to telomeric ssDNA, and inhibits

ATR-dependent signaling pathways. Depletion of POT1 leads to TIF formation, G-overhang

elongation, and activation of ATR-dependent cell cycle checkpoints (Lazzerini Denchi and de

Lange, 2007; Churikov and Price, 2008; Guo et al, 2007). One current hypothesis is that POT1

represses ATR-dependent signaling pathways by blocking the binding of RPA, and the

subsequent recruitment of ATRIP/ATR to ssDNA at telomeres (Lazzerini Denchi and de Lange,

2007).

Protein binding of the G-strand overhang appears to be the predominant form of telomere

capping in Saccharomyces cerevisiae, which do not form t-loops under normal circumstances.

S. cerevisiae have a trimeric RPA-like complex called Cdc13/Stn1/Ten1 (CST) which binds to

the 3’ overhang and plays a key role in telomere protection and length regulation (Gao et al,

2007). The CST complex was hypothesized to have been replaced by shelterin in higher

eukaryotes, however homologs of Stn1/Ten1 have recently been identified in multiple systems

(Martin et al, 2007; Miyake et al, 2009; Song et al, 2008; Surovtseva et al, 2009). Accumulating

evidence suggest that this type of shelterin independent, RPA-like complex also localizes to

ssDNA at telomeres and plays a role in telomere capping in most organisms. Arabidopsis

thaliana deficient in components of the CTC1/STN1/TEN1 complex show telomere length

heterogeneity, increased G-overhang signals, accumulation of extra-chromosomal circular

telomeric DNA, and increased end fusions involving subtelomeric sequences (Surovtseva et al,

2009; Song et al, 2008). SiRNA knockdown of human CTC1 results in increased telomere free

ends, formation of γH2AX foci in interphase cells, and increased single-strand G-rich DNA both

at the overhang and at internal sites (Surovtseva et al, 2009; Miyake et al, 2009).

As well as the shelterin and CST complexes, a number of DNA repair proteins have been

implicated in telomere biology. Many of these DNA repair factors accumulate at telomeres at

lower levels then the major telomere binding proteins, and localize to telomeres in a cell cycle-

specific manner. A role for some of these factors, such as the Werner and Bloom syndrome

5

helicases, in promoting replication of telomeres has been proposed (Crabbe et al, 2004; Sfeir et

al, 2009). Other factors such as the RAD51 paralog, RAD51D, may be required for the efficient

formation of t-loops (Tarsounas et al, 2004). In vitro experiments testing the ability of

immunodepleted human cell lysates to promote strand invasion of a linear telomeric substrate

with a 3’ overhang into a plasmid with telomeric repeats, suggests a role for RAD51, RAD52,

XRCC3, NBS1, RPA34 and ATR in t-loop formation (Verdun and Karlseder, 2006). The

mechanism of G-strand overhang production also likely requires a nuclease, whose identity is

currently unknown. Both the recruitment of HR and nucleolytic factors would likely require

activation of a DNA damage response, which may explain the observation that functional

telomeres in primary cells transiently activate a DNA damage response during G2, characterized

by the association of MRE11, NBS1 S343, RAD51, RAD52, XRCC3 and ATM S1981 with

telomeric DNA (Verdun et al, 2005; Verdun and Karlseder, 2006).

In addition to protein capping factors and higher order DNA structures, a growing body of

evidence also suggests a role for epigenetics in the regulation of mammalian telomere function.

The telomeric C-strand is transcribed by RNA polymerase II to generate noncoding transcripts,

known as Telomeric Repeat Containing RNAs (TERRA). TERRA has been detected in

mammals, zebrafish, and yeast, and can range in size from ∼100bp to >9kb in length (Azzalin et

al, 2007; Schoeftner and Blasco, 2008; Luke et al, 2008). TERRA associates with telomeres

in vivo, and can form G-quadruplex structures both on its own, and in conjunction with telomeric

ssDNA in vitro (Azzalin et al, 2007; Schoeftner and Blasco, 2008; Randall and Griffith, 2009;

Xu et al, 2008). Correlative analysis of TERRA expression and telomere length suggests that one

function of TERRA may be to inhibit telomere elongation by telomerase. However, siRNA

depletion of TERRA in human telomerase positive cells leads to an increase in telomere free

ends and telomeres with a FISH staining pattern resembling fragile sites (Deng et al, 2009).

Depletion of TERRA in human cells that do not rely on telomerase for telomere maintenance

results in increased TIF formation and reduced cell viability, suggesting TERRA also has

telomerase-independent affects on telomere capping (Deng et al, 2009). Studies in mice also

show an effect of the density of histone heterochromatic marks in telomeric and subtelomeric

sequences on telomere length and recombination (reviewed in Schoeftner and Blasco, 2009).

Although some of the potential mechanisms of capping appear to be redundant, interference with

any one of these mechanisms can affect telomere function, suggesting that they all have essential

6

roles. How all of these different factors are coordinated and contribute to telomere capping

remains to be elucidated.

1.1.2 Telomere Maintenance Mechanisms

The inability of DNA polymerase to fully replicate the terminal end of the lagging strand

coupled with the active processing required to generate the G-strand overhang, leads to

progressive telomere shortening of approximately 50-150 bp per population doubling in human

cells (Huffman et al, 2000; Martens et al, 2000). This shortening can be counteracted through the

activation or upregulation of a telomere maintenance mechanism. The primary mechanism of

maintaining telomere length relies on a multisubunit ribonucleoprotein complex called

telomerase. The minimal components of the enzyme that are required for catalytic activity are

the telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC)

(Weinrich et al, 1997). The template strand of TERC (AAUCCCAAUC) pairs with the end of

the 3’overhang, and then a single telomeric repeat is added per elongation step. Telomerase

preferentially elongates the shortest telomeres within a cell, and the addition of telomeric repeats

appears to be regulated in cis by telomere binding proteins, resulting in telomeres with a narrow

length distribution in telomerase positive cells (Britt-Compton et al, 2009; Smogorzewska and de

Lange, 2004). While widely expressed in single cellular organisms and some multicellular

organisms, telomerase expression is undetectable in most human somatic cells (Kim et al, 1994).

Instead, in humans, telomerase expression appears to be largely restricted to brief periods early

in development, and to germ-line and stem cell compartments (Kim et al, 1994; Wright et al,

1996).

Lack of telomerase expression in human somatic cells contributes to an age related decline in

average telomere lengths, as well as the progressive telomere shortening of cells grown in culture

(Lindsey et al, 1991; Vaziri and Benchimol, 1998). In vitro experiments have shown that human

primary cells can proliferate a limited number of times in culture, and then enter a stage of

permanent arrest referred to as replicative senescence or mortality stage 1 (M1) (Figure 1-2)

(Hayflick, 1965). Senescence of cells in culture can also be induced by oncogene overexpression,

exposure to DNA damaging agents, or inadequate culture conditions, however these

phenomenon appear distinct from M1, and are instead referred to as stasis (Serrano et al, 1997;

Robles and Adami, 1998; Sherr and DePinho, 2000; Drayton and Peters, 2002). As populations

7

of cells approach replicative senescence, cells begin to appear with telomeres that have less the

100bp of C-strand DNA and are below the limit of detection with FISH (Zou et al, 2004). These

critically short telomeres associate with DNA repair foci, suggesting that they have activated a

DNA damage response, leading to activation cell cycle checkpoints and cellular senescence (Zou

et al, 2004). Supporting the idea that telomere shortening is the driving force behind replicative

senescence, expression of telomerase can extend cellular lifespan beyond this M1 point (Vaziri

and Benchimol, 1998; Bodnar et al, 1998).

Figure 1-2 Model of the telomere length hypothesis of cellular senescence and

immortalization. Repeated cellular divisions leads to progressive loss of telomeric DNA in human somatic cells. Cells

begin to accumulate critically short telomeres that trigger checkpoint activation at mortality stage one

(M1). Bypass of cellular checkpoints allows cells to continue dividing, until a period of massive genomic

instability referred to as crisis or mortality stage (M2) is reached. Emergence from crisis requires

activation or upregulation of either telomerase, or the Alternative Lengthening of Telomeres (ALT)

pathway.

Impairment of cell cycle checkpoints allows cells to continue cycling beyond M1, until a period

known as mortality stage 2 (M2) or crisis is reached, at which point widespread cell death is

triggered (Figure 1-2). During the period of between M1 and M2 telomeres continue to shorten

and cells accumulate dicentric chromosomes and telomere associations (Zou et al, 2009).

8

Telomere associations differ from true fusions in that they are not mediated by a ligase IV

dependent end-joining mechanism and a constriction indicating the physical end of both

chromosomes is still visible (Zou et al, 2009). The existence of both telomere associations and

fusions suggests that either dysfunctional telomeres may be dealt with by different pathways, or

that end fusions may often be incomplete, potentially due to a suppressive effect of residual

telomere sequence and binding proteins on DNA repair. This would lead to a model where there

are different levels or subtypes of telomere uncapping, one level at which telomeres are

sufficiently uncapped to stimulate TIF formation but retain sufficient capping abilities to

suppress fusions, and another more fully uncapped state which results in TIFs and fusions.

Supporting the idea that not all uncapped telomeres are equal, metaphase spreads of

immortalized and cancerous cells frequently have telomeres that are TIF positive, but are not

involved in fusions or associations with other telomeres (Cesare et al, 2009).

Cellular emergence from M2 requires the activation or upregulation of a telomere maintenance

mechanism. The majority of cells rely on telomerase, however ∼30% of in vitro immortalized

cells rely on a telomerase independent form of telomere maintenance called the Alternative

Lengthening of Telomeres (ALT) pathway (Bryan et al, 1995). The ALT pathway is also active

in ∼10% of human cancers, but as of yet has not been shown to be active in a primary cell setting

(Bryan et al, 1997). The underlying mechanism of ALT is not fully understood, making it

impossible to know whether ALT represents a single or multiple pathways.

The first ∼1.9kb of human telomere arrays begins with the consensus telomere repeat

(TTAGGG), interspersed with variants of this sequence including but not limited to TGAGGG,

TCAGGG, TTGGGG (Allshire et al, 1989). Monitoring sequence changes in this region of

chromosomes 12 and 16 in clones grown up from single cells shows no mutations in primary

MRC-5 and WI38 cells, only one mutation in 97 clones from a SV40 immortalized pre-crisis cell

line, and no mutations in HT1080 telomerase positive fibrosarcoma cells (Varley et al, 2002). In

contrast, multiple ALT cell lines show highly elevated frequencies of sequence changes,

including both loss of the progenitor telomere at discrete fusion points where it is replaced by a

different telomere array, as well as intra-allelic changes which may be due to insertions,

deletions, and base-changes (Varley el al, 2002). This suggests that telomeric DNA within ALT

is highly unstable relative to telomerase positive and primary cells.

9

Instability in ALT cells may not be limited solely to telomeric sequences. A comparison of

genomic single nucleotide polymorphisms in liposarcoma tumors that have activated ALT,

telomerase, or no apparent telomere maintenance mechanism showed that ALT tumors tended to

have higher levels of genomic instability, more frequent loss of heterozygosity, and frequent

deletion of 1q32.2-q44 (Johnson et al, 2007). Additionally, the MS32 minisatellite located at

1q42.3 also shows frequencies of instability that are on average ∼55 fold greater in 15 different

ALT cell lines then non-ALT cell lines (Jeyapalan et al, 2005). This increase in MS32 instability

was also observed in 3 out of 8 ALT sarcoma samples, but ALT activation did not correlate with

instability at 4 other minisatellites (Jeyapalan et al, 2005). Increased instability at telomeres and

other loci in ALT cells suggests that ALT may be an inferior mechanism of maintaining

telomeres, ALT activation may be caused by mutations that lead to an overall increase in

genomic instability, and/or ALT may be activated after a longer period in crisis.

As the genetic details of ALT are undefined, cells are characterized as being ALT based on the

presence of certain characteristics. A common characteristic of ALT cells is that they contain

long and heterogeneous telomeric DNAs. While telomeres in primary and telomerase positive

cells do not generally exceed 10-15kb in length, telomeric DNA isolated from ALT cells

typically range from <2kb to >50kb when assessed by southern blotting (Bryan et al, 1995).

Using the presence of hypervariable telomeric DNA lengths as a marker of ALT, cell fusion

studies were carried out wherein ALT immortalized human cells were fused to either primary

fibroblasts or telomerase positive immortalized human cells. Fusion of the GM847 ALT cells

with any of 3 different primary diploid fibroblast lines results in loss of abnormally long

telomeric DNA and induction of cellular senescence (Perrem et al, 1999). When GM847 cells

were fused to telomerase positive cells from the same immortalization complementation group,

all clones lost abnormally long telomeric DNA, and 2 out of 9 clones showed a transient growth

arrest (Perrem et al, 1999). These experiments suggest that ALT may arise from one or more

recessive mutations, and that both primary and telomerase positive cells contain repressors of

ALT.

A second characteristic of ALT cells is that they contain high levels of Extra-Chromosomal

Telomeric Repeat (ECTR) DNA, which is both linear and circular, single-stranded and double-

stranded (Tokutake et al, 1998; Ogino et al, 1998; Wang et al, 2004a; Cesare and Griffith, 2004;

Nabetani and Ishikawa, 2009). Electron microscopic analysis of DNA fractions enriched for

10

telomeric repeats showed the presence of circular molecules ranging in size from <2kb to >50kb,

with ∼60% of circles being <6kb (Cesare and Griffith, 2004). T-loop structures are also present

in ALT cells, with loop portions ranging in size from 0.5-70kb, but ∼75% of loops being <5kb,

and the total length of the loop and tail ranging from 0.9-79 kb (Cesare and Griffith, 2004). The

similar size of most free circular DNA molecules and the loop portion of t-loops support a model

where ECTR circular DNA is generated through aberrant resolution of t-loops (Figure 1-3 A).

T-loops contain a partial or full Holliday junction at their base, which could be resolved to

generate a free ECTR DNA circle and a significantly shorter telomere. Monitoring of the length

of a single tagged telomere shows that ALT telomeres shorten at a rate of ∼50 bp per population

doubling, but are also subject to rapid shortening and elongation events, explaining the wide

variability of telomere length present within a given cell (Murnane et al, 1994; Henson et al,

2002).

A)

B)

Figure 1-3. Potential mechanisms of extra-chromosomal telomeric repeat (ECTR) DNA

production in ALT

A) T-loops may be aberrantly resolved to generate a short telomere and circular or linear piece of ECTR

DNA. B) Unrepaired double-strand breaks within telomeres can generate a short telomere and a linear

piece of ECTR DNA. Ligation of linear ECTR DNA can also result in production of circular ECTR.

11

Ligation of linear ECTR DNA is an alternate mechanism of generating circular ECTR DNA. The

mechanism of linear ECTR DNA production is currently unknown, but could occur during

aberrant resolution of t-loops, introduction of double-strand breaks within telomeres or at

telomere proximal sequences, or endonucleolytic cleavage of either telomeres or circular ECTR

DNA (Figure 1-3 B). Alternately linear ECTR DNA could be a byproduct of replication and

recombination reactions among existing ECTR species. Study of the repair of I-Sce1 induced

double-strand breaks located either 3kb or 100kb from a telomere, shows an increase in large

deletions, terminal deletions, and gross chromosomal rearrangements at telomere proximal

breaks relative to other random interstitial sites, with more pronounced effects at the 3kb site

(Zschenker et al, 2009; Kulkarni et al, 2010). This suggests that telomeres can negatively effect

the ability of cells to repair double-strand breaks. Whether the magnitude of the affect of

telomeres on DNA repair is proportional to telomere length is unclear, but it is possible that long

telomeres present within ALT create an environment within the telomeric or subtelomeric

sequence that is abnormally difficult to repair, leading to more frequent rapid deletions of

telomere sequence arising from double-strand breaks. Interestingly, when U2OS cells were

transfected with a non-targeting plasmid and clones that had stably integrated the plasmid where

selected, 29 out of 30 clones had integration sites at close proximity to the telomere as assessed

by FISH (Jegou et al, 2009). The presence of a double-strand break results in an increase in the

frequency of viral integrations at that site, suggesting that one explanation for the integration of

the plasmid near telomeres would be the presence of double-strand breaks (Bill and Summers,

2004).

A third characteristic of ALT cells is the presence of promyelocytic leukemia (PML) nuclear

bodies that colocalize with telomeric DNA and shelterin proteins, termed ALT-associated PML

Bodies (APBs) (Yeager et al, 1999). APBs are not normally observed in telomerase positive or

primary cells, and arise at around the same time as long and heterogeneous telomeric DNAs,

suggesting the two phenomena are related (Yeager et al, 1999). In addition to telomeric factors,

APBs also colocalize with proteins implicated in DNA repair, replication and recombination,

including but not limited to RPA, RAD51, RAD52, RAD9, RAD1, HUS1, RAD17 and BLM

(Yeager et al, 1999; Stavropoulos et al, 2002; Nabetani et al, 2004). APBs accumulate during the

G2 phase of the cell cycle and disruption of ALT telomere maintenance leads to reduced

numbers of APBs (Grobelny et al, 2000; Potts and Yu, 2007). These observations have led to the

12

hypothesis that APBs are sites for telomere elongation and deletion events, although direct

evidence supporting this hypothesis is currently lacking, and the role of APBs within ALT

remains a subject of debate.

The final characteristic of ALT telomere maintenance is an increase in telomeric recombination.

The frequency of homology directed repair at sites of I-Sce1 induced double-strand breaks at an

intra-chromosomal site proximal and distal to telomeres does not significantly differ between

ALT and non-ALT cells. This suggests that homology directed repair at non-telomeric sites is

not elevated in ALT cells (Bechter et al, 2003). Studies on the frequencies of spontaneous

homologous recombination events in ALT and non-ALT cells have not been carried out,

however the frequency of genomic SCEs is not elevated in ALT cells relative to telomerase

positive cells (Londoño-Vallejo et al, 2004). The first evidence of increased recombination

between ALT telomeres came from experiments where a tag was inserted into the telomeric

repeats of a single telomere in both an ALT, and a telomerase positive cell line (Dunham et al,

2000). Within the ALT cell line the tag eventually spread to telomeres on other chromosomes, a

phenomenon which did not occur within the telomerase positive cell line (Dunham et al, 2000).

When the tag was placed in the subtelomeric region of an ALT cell, it was not copied to multiple

chromosomes. This result suggested a model where one ALT telomere can invade another, using

the other telomere as a template during replication. Alternately, the tagged sequence may move

through events involving ECTR DNA.

The second line of evidence suggesting telomeric recombination is no longer inhibited in ALT

cells, was provided by chromosome orientation fluorescent in situ hybridization experiments

(CO-FISH). During CO-FISH experiments cells are grown in BrdU/BrdC for one round of

replication, which increases the sensitivity of the newly synthesized strand to ultraviolet induced

damage, allowing for its selective degradation. Using FISH probes specific for either the G

and/or C strand, telomeres that have undergone an exchange within the telomeric sequence can

be identified. Exchanges are hypothesized to be due to crossover reactions between sister

telomeres, and are referred to as telomere sister chromatid exchanges (T-SCEs). If exchanges are

asymmetric they will result in elongation of one telomere and shortening of the other, however to

date there is no direct evidence that a T-SCE results in a net change in telomere length.

Additionally, T-SCEs may also be caused by exchanges between non-sister telomeres, or

telomeres and ECTR DNA. T-SCEs occur at frequencies of 28-280/100 metaphases in different

13

ALT cell lines, but are rarely if ever observed in telomerase positive and primary human cells

(Londoño-Vallejo et al, 2004).

The presence of ECTR DNA circles is frequently interpreted as measure of intra-telomeric

recombination, as circles may arise through inappropriate resolution of the t-loop junction. While

telomeric circles are readily detectable by 2D gel analysis of DNA from cells that utilize the

ALT pathway, they are typically undetectable or present at only very low levels in telomerase

positive and primary cells (Wang et al, 2004a; Cesare and Griffith, 2004). Additional evidence of

intra-telomeric recombination within ALT cells was provided by experiments using a telomere

integrated tag encoding a splice acceptor site upstream of a red fluorescent protein (RFP) open

reading frame, followed by a CMV promoter and splice donor site. When present only once

within the telomere, the CMV promoter is unable to drive RFP expression, however if the tag is

repeated the promoter will be upstream of RFP, and following splicing of the transcript, RFP will

be translated and cells will fluoresce. When the tag was incorporated into ALT cells it was

copied multiple times within a single telomere resulting in RFP expression (Muntoni et al, 2009).

Integration of the tag into telomerase positive cells did not result in RFP expression. Potential

mechanisms of intra-telomeric duplication include t-loop mediated rolling circle replication,

copying of the tag between sister telomeres, and unequal T-SCEs.

There are multiple mechanisms that may contribute to the rapid elongation of ALT telomeres,

including rolling circle replication involving either an intra-telomeric loop or ECTR circular

DNA, break induced replication between telomeres, recombination between a telomere and

linear ECTR DNA, or unequal exchanges between telomeres (Figure 1-4). In addition to

recombination-based mechanisms, ligation of ECTR DNA to telomeres may also play a role. In

yeast, telomerase inactivation results in telomere shortening and telomere-induced senescence.

Rare survivors primarily rely on recombination-based Rad52p dependent pathways, however if

elongated telomeres are present during before senescence, a Rad52-independent pathway that

produces heterogeneous and hypervariable telomeres can be used (Grandin and Charbonneau,

2009). Genetic analysis suggests that this pathway does not function via recombination, single-

strand annealing, nonhomologous end-joining or break induced replication. This pathway does

require the Rad1-Rad10 endonuclease, the replication factor C component Egl1, and the

Mre11/Rad50/Xrs2 complex, and has been proposed to function via microhomology-mediated

end joining between telomeres and extra-chromosomal telomeric repeat DNA generated during

14

telomere rapid deletion events (Grandin and Charbonneau, 2009). Whether or not an analogous

pathway functions within human ALT cells is currently unknown.

Figure 1-4. Potential mechanisms of recombination based telomere elongation in ALT cells

A) Both the 3’ and 5’ ends of the telomere pair with their complementary strands at the base of the t-loop

forming a 4 strand rolling circle substrate, allowing both strands to be extended simultaneously.

Alternately, the newly synthesized DNA from the 3’ invading strand may be continuously displaced and

subsequently converted into a double-strand product. B) The 3’ end of the telomere pairs with a

complimentary single-strand ECTR DNA circle, allowing for rolling circle replication. The newly

synthesized telomeric DNA would be subsequently converted into a double-strand product. Alternately

the telomeric end could form a 4 strand rolling circle through invasion of double-strand ECTR DNA

circle, allowing for simultaneous extension of both strands. C) The 3’ end of one telomere invades

another, and then is extended through break induced replication. D) The 3’ end of the telomere invades

double-strand ECTR DNA forming a Holliday junction that is resolved in a way that results in extension

of the telomere, and shortening of the ECTR DNA. Unlike A-C which involve replication and a net

increase in telomeric DNA, in D and E there is no overall change in total telomeric DNA content E) The

3’ end of the telomere invades another telomere at a more proximal point, forming a Holliday junction

which is resolved to yield one longer and one shorter telomere.

A) B)

C)

D)

E)

15

1.1.3 Telomeres and Disease

The role of telomere dysfunction in human disease and aging is a subject of great research

interest, but has proven to be technically challenging to investigate because in most model

systems, telomeres do not appear to play a regulating cellular or organismal lifespan. Mouse

models most frequently used to study human disease have long telomeres, express telomerase in

most tissues, and do not normally rely on telomeres as a mechanism to count cell divisions

(Blasco et al, 1997; Wright and Shay, 2000). Supporting a different role for telomeres in humans

vs model organisms, telomerase mutations resulting in a null phenotype have never been

identified in humans, but telomerase null mice and Caenorhabditis elegans are viable for up to 6

generations, while telomerase null Arabidopsis thaliana are viable up to 10 generations (Blasco

et al, 1997; Riha et al, 2001). The ability of these systems to remain viable for multiple

generations without telomerase may be due to combination of factors including the initial

presence of extended telomere sequences, the ability to employ telomerase independent

pathways to extend telomeres, only small losses of telomeric DNA per cell division, preferential

inheritance of rare germ cells with long telomeres, or increased tolerance to genomic instability

(Riha et al, 2001; Cheung et al, 2006).

While it typically takes several generations for telomerase null model organisms to exhibit

phenotypes, mutations that affect telomerase activity for a single generation can result in human

disease. These rare disorders currently provide some of the best evidence that telomere

dysfunction can directly impact human health. Dyskeratosis Congenita (DC) is a rare inherited

bone marrow failure syndrome diagnostically characterized by skin hyperpigmentation, nail

dystrophy and mucosal leukoplakia (Knight et al, 1998). In addition, pulmonary fibrosis,

premature grey hair, liver disease, short stature, microcephaly and developmental delay are

observed in some individuals. Haematological abnormalities are extremely common in DC, with

over 85% of patients experiencing a cytopenia of a single lineage, and 76% of patients

developing pancytopenia by a median age of 10 (Garcia et al, 2007). Bone marrow failure is the

leading cause of death in DC, followed by pulmonary disease and cancer (Knight et al, 1998).

Head and neck squamous cell carcinomas are the most frequent cancer in DKC, occurring in

∼40% of patients (Alter et al, 2009). The ratio of observed to expected cases of myelodysplastic

syndrome are also extremely elevated in DKC (>2500 fold), as are cases of tongue cancer

(>1100 fold), and acute myeloid leukemia (>200 fold) (Alter el al, 2009).

16

DC is genetically heterogeneous, displaying X-linked recessive, autosomal dominant, and

autosomal recessive inheritance. X-linked DC is caused by mutations in DKC1, whose gene

product dyskerin, associates with the telomerase RNA component and is a component of the

telomerase holoenzyme (Heiss et al, 1998; Mitchell et al, 1999). Autosomal forms of the disease

are due to mutations in genes encoding the telomerase reverse transcriptase (TERT), RNA

component (TERC), additional components of the telomerase holoenzyme (NHP2, NOP10), or

the shelterin component TIN2 (Marrone et al, 2007; Vulliamy et al, 2001; Vulliamy et al, 2008;

Walne et al, 2007; Savage et al, 2008). Autosomal dominant forms of the disease are typically

due to mutations in TERC or TERT, and function via haploinsufficiency and not a dominant

negative mechanism (Vulliamy et al, 2001; Marrone et al, 2004; Armanios et al, 2005). In vivo

telomerase reconstitution experiments suggest that mutations that cause a <50% reduction in

telomerase activity are sufficient to cause disease (Marrone et al, 2007).

Telomeres in DC patients are extremely short, with average telomere lengths in leukocytes

following below the first percentile relative to age-matched controls (Alter et al, 2007). The

spectrum of genes implicated in DC combined with this short telomere phenotype, implicate

telomere shortening as the proximal cause of the disease. The high incidence of bone marrow

failure in DC suggests that the haematopoietic system is particularly vulnerable to changes in

telomerase activity or telomere uncapping. It should be noted that while the majority of

measurements of telomere length focus on the average telomere length, the shortest telomeres

appear to be the key driving force in both replicative senescence and chromosome fusions in

situations of spontaneous genomic instability (Zou et al, 2004; Pampalona et al, 2010).

Critically short telomeres are usually several kb shorter then other telomeres within the cell, and

are undetectable by FISH, suggesting that they have undergone one or more rapid deletion events

at some point in their replicative history. While telomerase may preferentially elongate the

shortest telomeres, it is estimated that in human cells a maximum of 544bp of telomeric DNA

can be added per population doubling (Britt-Compton et al, 2009). As telomeres shorten, they

may become more prone to stochastic changes in telomere length due to a partial capping defect

caused by reduced numbers of shelterin components associated with telomeres. In this model

even modest changes in telomerase activity can result in a situation where progressive decreases

in average telomere length leads to increased production of critically short telomeres, which

cannot be adequately extended by telomerase, ultimately leading to cell death or senescence.

17

Examination of the ratio of telomere lengths on the long and short arms of each chromosome,

which is usually stable but will change if one telomere is rapidly shortened or elongated, shows

that cells from a DC patient are approximately twice as likely to have chromosomes with a >5

fold change in the telomere length ratio as cells from an non-affected relative (Morrish and

Greider, 2009). The mechanism driving stochastic changes in telomere length is unknown,

however is hypothesized to be a result of telomeric recombination.

In addition to DC, mutations in TERC have been identified in a small number of patients with

aplastic anaemia (2/155) and myelodysplatic syndrome (1/55) (Yamaguchi et al, 2003). TERT

mutations have also been identified in aplastic anaemia patients (7/200), suggesting that other

bone marrow failure syndromes may be directly caused by telomere dysfunction (Yamaguchi et

al, 2005). Recently, TERC and TERT mutations have also been identified in patients with

familial idiopathic pulmonary fibrosis at frequencies of approximately 1.4% (1/73) and 6.8%

(5/73) of patients, respectively (Armanios et al, 2007). Average telomere lengths in lymphocytes

in probands as well as asymptomatic carriers were below the 10th percentile relative to age-

matched controls, suggesting that reduced proliferative potential may contribute to this disease

(Armanios et al, 2007).

1.2 Fanconi Anaemia (FA)

1.2.1 The FA Clinical Phenotype

Fanconi anaemia (FA) is a genetically and phenotypically heterogeneous disorder characterized

by progressive bone marrow failure, increased cancer susceptibility, and congenital

abnormalities. As the clinical phenotype of FA can vary, confirming diagnosis relies on detection

of increased chromosome breakage in response to treatment with DNA crosslinking agents, a

class of mutagens that FA cells are hypersensitive to (Auerbach et al, 1989). Analysis of 754 FA

cases collected by the International Fanconi Anemia Registry between 1982 and 2003, showed

that 90% of patients experienced bone marrow failure by age 40, and that the median survival

age of patients is 24 (Kutler et al, 2003). Myelodysplastic syndrome and/or acute myeloid

leukemia are also extremely common in FA, with a cumulative incidence of 33% by age 40

(Kutler et al, 2003). In addition to haematological malignancy, FA patients are also prone to

developing solid tumors, with a cumulative incidence of 28% by age 40 (Kutler et al, 2003). The

majority of tumors in FA patients are squamous cell carcinoma of the head and neck or ano-

18

genital region, followed by liver, brain, and renal cancers. The ratio of observed to expected

cancers in FA is highest for leukemia (785x), liver (386x), head and neck (706x), esophageal

(2362x), cervical (179x), and vulvar (4317x) neoplasms (Rosenberg et al, 2003).

Major congenital abnormalities are present in approximately 2/3 of FA patients, the most

common of which are radial ray abnormalities, gastrointestinal malformations and abnormalities

of the central nervous system (Giampietro et al, 1997; Giampietro et al, 1993). In addition to

major malformations, minor abnormalities frequently observed include skin hyper and hypo-

pigmentation, microcephaly, micropthalmia, and height and weights around the 5th percentile

(Auerbach, 2009). FA patients have reduced fertility, with males displaying evidence of

hypoplastic gonads and abnormal spermatogenesis (Auerbach, 2009). There is no strong

genotype/phenotype correlation connecting the congenital malformations, as in an analysis of 45

groups of FA siblings, 12 sets contained siblings with and without malformations, and 12 sets

contained siblings with malformations of differing severity (Giampietro et al, 1993).

Additionally, monozygotic FA twins have been identified both with and without malformations,

and different malformations (Auerbach, 2009). This suggests that although FA gene mutations

drastically increase the probability of developmental anomalies, they may arise through a

stochastic process. An additional factor that may influence FA patient phenotypes is somatic

mosaicism, caused by spontaneous reversion of inherited mutations or acquisition of a secondary

compensatory mutation. Approximately 25% of FA patients have peripheral lymphocyte

populations that are ~25% corrected, and 10% of patients have lymphocyte populations that are

~50% corrected (Auerbach, 2009).

The underlying problem driving the bone marrow failure in FA is currently unknown, however

the dominant hypothesis is that it is direct result of exhaustion of haematopoietic stem cells. FA

cells exhibit increased spontaneous genomic instability, visible in examination of metaphase

spreads which show increased frequency of breaks, fusions, gaps and radials (Schroeder and

Kurth, 1971; Schroeder and German, 1974). Unlike wild-type cells that exhibit a small and

constant number of chromosomal aberrations when grown under different oxygen tensions (2

breaks per 100 metaphases), the number of aberrations in FA cells increases dramatically with

increased oxygen levels (80 breaks per 100 metaphases at 40% oxygen) (Joenge et al, 1981). An

increasing body of evidence suggests that proteins implicated in FA play important roles in

certain DNA repair pathways. An intrinsic inability of FA cells to adequately deal with

19

endogenous DNA lesions may play a role in the bone marrow failure, developmental

abnormalities, and high cancer incidence observed in FA patients, as cells with

unrepaired/misrepaired damage may be more prone to undergo apoptosis or senescence, or if

they escape this fate, will have increased levels of genomic instability which may help to drive

oncogenic progression.

1.2.2 FA and Telomere Maintenance

One potential source of endogenous DNA damage in FA cells are dysfunctional telomeres,

which may arise due to defects in telomere capping, frequent rapid losses of telomeric DNA,

reduced telomerase activity, or as a secondary affect of excessive proliferation of haematopoietic

cells. FA shares several clinical characteristics with DC, including extremely high frequencies of

bone failure, myelodysplastic syndrome, acute myeloid leukemia, and head and neck squamous

cell carcinomas. Initial telomere studies in FA peripheral blood mononuclear cells examined

average telomere lengths by southern blotting in 6 FA patients, and found that in 4 patients

telomeres were 1.2 – 2.2 kb shorter then age-matched controls (Ball et al, 1998). However the

other 2 patients analyzed in this study had normal average telomere lengths, despite both having

bone marrow failure (Ball et al, 1998). A subsequent larger study on blood samples from 45 FA

patients showed that telomeres were on average 1.75kb shorter then controls, and that telomeres

in patients that had developed one or more cytopenias were approximately 0.94kb shorter then

patients without a cytopenia (Leteurtre et al, 1999). This work was further extended to show that

patients with cytopenias had an increased rate of annual telomere shortening (>200bp/year), and

that a high rate of annual telomere shortening could serve as a prognostic indicator of

progression towards a more severe haematological disease (Li et al, 2003). Whether or not the

observed telomere shortening is a cause, or a consequence of haematopoietic failure or stress is

not apparent from these studies.

A more recent direct comparison of average telomere lengths in populations of peripheral blood

cells from DC and FA patients using flow cytometric FISH measurements has shown that the

pattern and extent of telomere shortening differs significantly between the two diseases (Alter et

al, 2007). While telomeres do tend to be shorter then average in FA cells, this is mainly observed

in the granulocyte population, whereas telomeres in DC tended to be extremely short (<1st

percentile) in all lineages tested. Looking at the occurrence of extremely short telomeres, only 7

20

of 16 FA patients met the criteria in one or more cell types. Of these 7 patients only 3 had

extremely short telomeres in more then 3 lineages, however 2 of these patients had received prior

radiation therapy. This suggests that if telomere dysfunction is involved in FA, the mechanism

driving it is likely to differ from DC. Supporting this idea, telomerase activity does not appear to

be decreased when tested in blood samples from FA patients, and mutations in TERC have not

been identified (Leteurtre et al, 1999; Calado et al, 2004).

The above studies focused on average telomere length, however telomere dysfunction causing

improper capping or increased telomere rapid deletions would not be easily detected using this

approach. One study that looked at individual telomere lengths in metaphase spreads of FA

patient lymphocytes found that there was an increase in the frequency of chromosome ends

without detectable telomere signals, whereby in FA cells 0.26% of chromosomes had a

chromosome end with an undetectable telomere via FISH, while in controls this value was 0.15%

(Callén et al, 2002). In this same study, an average of 7.8 extra-chromosomal telomeric signals

per cell was observed in FA cells, but only 2.3 signals per cell in controls, and there was a

greater then 10 fold increase in chromosome end fusions (Callén et al, 2002). However the

average telomere lengths in FA cells (3.95 kb) was significantly shorter then controls (4.63 kb),

which may have resulted in a situation where telomeres are becoming uncapped and more prone

to rapid deletion events due to telomere shortening, and not as a direct result of FA mutations. A

study examining individual telomere lengths by FISH in FA fibroblasts with longer telomere

lengths (10.5 ± 4.2 kb, 9.7 ± 5.2kb) failed to show any increase in chromosome ends without a

detectable sequence or end fusions, and did not cause a shift in telomere length distribution

consistent with rapid loss of telomeric sequence (Franco et al, 2004). The major aim of this study

is to clarify what role, if any, the FA pathway plays in telomere maintenance.

1.2.3 The FA Pathway

There are currently 12 identified genes which when mutated in humans give rise to FA (FANCA,

B, C, D1/BRCA2, D2, E, F, G, I, J/BACH1/BRIP1, L, N/PALB2) (de Winter and Joenje, 2009).

With the exception of FANCB, which demonstrates X-linked inheritance and has only been

identified in males, all other FA mutations function in an autosomal recessive manner and are

found at approximately equal frequencies in male and female patients (Kutler et al, 2003;

Auerbach, 2009). Patients are divided into complementation groups based on their gene

21

mutation, analysis of 681 FA with known complementation groups shows the following

distribution of patients: FA-A=411 (60.4%); FA-B=10 (1.5%); FA-C=108 (15.9%); FA-D1=20

(2.9%); FA-D2=16 (2.3%); FA-E=9 (1.3%); FA-F=16 (2.3%); FA-G=67 (9.8%); FA-I=6 (0.9%);

FA-J=13 (1.9%); FA-L=2 (0.3%); FA-N=3 (0.4%) (Auerbach, 2009). All of the FA genes

identified to date encode proteins that appear to function within a common pathway (Figure 1-6),

although many FA proteins have additional roles outside of this pathway.

The first FA genes identified all encoded proteins that are components of a large complex

referred to as the FA core complex (FANCA, C, E, F, G) (Strathdee et al, 1992; Lo Ten Foe et al,

1996; de Winter et al, 1998; de Winter et al, 2000a; de Winter et al, 2000b).

Immunoprecipitation of FANCA coupled with mass spectroscopy has been used to identify

additional components of this complex, referred to as FANCA-associated polypeptides (FAAPs)

(Meetei et al, 2003). Mutations in FAAP43, FAAP95, and FAAP250 were subsequently

identified in FA patients, and these proteins were renamed FANCL, B and M, respectively

(Meetei et al, 2003; Meetei et al, 2004a; Meetei et al, 2005). Two additional FAAPs, FAAP24

and FAAP100 also appear to be important for core complex function, however, to date patient

mutations in these genes have not been identified (Ciccia et al, 2007; Ling et al, 2007).

Determining the role of the FA core complex was initially hampered by the lack of known

functional domains present in the first five protein components identified. However the

discovery of FANCD2, a protein whose monoubiquitination is dependent on the expression of

FA core complex members, suggested a function for the core complex in regulating

ubiquitination (Timmers et al, 2001; Garcia-Higuera et al, 2001). FANCD2 monoubiquitination

is required for its association with chromatin and assembly into nuclear foci with other DNA

repair factors (Garcia-Higuera et al, 2001). FANCI was recently identified as a second protein

that forms nuclear foci and binds to chromatin following monoubiquitination by the FA core

complex (Smogorzewska et al, 2007; Sims et al, 2007). Monoubiquitination of FANCD2 and

FANCI appears to be carried out by FANCL, which has E3 ubiquitin ligase activity, and, with

the exception of worms, is present with FANCD2 and FANCI throughout evolution (Figure 1-5)

(Meetei et al, 2004b). Additional identified components of this pathway include UBE2T, which

acts as the E2 ubiquitin activating enzyme, and USP1 which promotes deubiquitination of

FANCD2 (Machida et al, 2006; Nijman et al, 2005).

22

Unlike the other FA core complex components which are essential for monoubiquitination,

FANCM appears to play more of an accessory role, wherein it promotes the association of the

FA core complex with chromatin and subsequent monoubiquitination of FANCD2

monoubiquitination, but is not absolutely required for these activities (Bakker et al, 2009). A

mouse FANCM model also reveals some novel phenotypes not shared with the other FA core

complex mouse models, including an underrepresentation of FANCM deficient female mice, and

an increase in the frequency of spontaneous Sister Chromatid Exchange events (SCEs) (Bakker

et al, 2009). The only FANCM patient identified to date also has biallelic FANCA mutations,

making the human FANCM phenotype unclear (Singh et al, 2009). Increased SCEs have also

been observed in FANCM depleted human cells, but not other FA cell types, and likely relate to

the ability of FANCM to recruit the Blooms syndrome complex (BLM/TopoIIIα/RMI1/RMI2) to

sites of stalled or collapsed replication forks (Deans and West, 2009).

Figure 1-5. Overview of FA pathway conservation in eukaryotes Many of the FA core complex components (blue) do not appear to conserved in simple eukaryotes with

the exception of FANCL and FANCM. FANCL has E3 ubiquitin ligase activity and is normally present

with FANCD2 and FANCI. FANCM is related to the archael DNA repair protein Hef, and has additional

roles outside of the FA pathway. Diagram modified from Zhang et al, 2009.

23

During normal replication and in response to treatment with exogenous DNA damaging agents

including ionizing radiation, UVC, interstrand crosslinking agents, and replication fork stalling

agents, FANCD2 and FANCI are monoubiquitinated in a core complex dependent manner at

lysine 561 and 523, respectively (Garcia-Higuera et al, 2001; Smogorzewska et al, 2007; Sims et

al, 2007). FANCD2 and FANCI protein stability and monoubiquitination appear interdependent,

and coimmunoprecipitation experiments suggest that the endogenous forms of these proteins

interact weakly or transiently in vivo (Sims et al, 2007). Human FANCI encodes a 150 kDa

protein that shares low overall sequence similarity (~20%) with the 155kDa FANCD2 protein,

however the region surrounding the monoubiquitination site shows approximately 40% similarity

(Timmers et al, 2001; Sims et al, 2007; Smogorzewska et al, 2007). Additionally, the interior of

both proteins contain leucine rich sequences predicted to form helical hairpin structures, similar

to what has been observed in crystal structures of FANCE and FANCF (Smogorzewska et al,

2007; Nookala et al, 2007; Kowal et al, 2007). The C-terminus of both FANCI also contains a

putative EDGE domain similar to what has been previously identified in FANCD2. In FANCD2

the EDGE domain is required for complementation of the interstrand crosslinker sensitivity

phenotype, but is not involved in FANCD2 monoubiquitination or foci formation (Montes de

Oca et al, 2005). These structural similarities between FANCD2 and FANCI strongly suggest

that they both evolved from a common ancestral gene.

Approximately 5% of FA patients carry mutations in genes that do not affect FANCD2 or

FANCI expression or monoubiquitination. Mutations in the BRCA1 Associated C-terminal

Helicase (BACH1) are causal in the FANCJ complementation group (Levitus et al, 2005; Litman

et al, 2005). Biallelic mutations in BRCA2 or its binding partner PALB2 are implicated in the

FANCD1 and FANCN complementation groups, however the clinical phenotype in these groups

differs significantly from other FA groups (Howlett et al, 2002; Reid et al, 2007). Unlike other

FA patients, the first adverse clinical effect observed in FANCD1 and FANCN patients typically

is an early childhood solid tumors (Offit et al, 2003; Reid et al, 2007). The high frequency of

Wilms tumor, medullablastoma, and neuroblastoma in FANCD1 and FANCN patients often

leads to mortality during early childhood, and may be a consequence of roles for FANCD1 and

FANCN outside of the FA pathway. Figure 1-6 shows a model of the activation and recruitment

of FA pathway components in response to DNA damage.

24

Figure 1-6. Model of FA pathway activation

FANCM translocates along the DNA until it encounters an interstrand crosslink (upper left). The

FA core complex is recruited to the site of damage (upper right). The FA core complex together

with an E1 ubiquitin activating enzyme and UBET, monoubiquitinate FANCD2 and FANCI.

Monoubiquitinated FANCD2 and FANCI assemble at the site of damage and interact with other

downstream FA proteins (FANCJ/BACH1, FANCD1/BRCA2, FANCN/PALB2) and DNA

repair proteins.

1.2.4 The Role of FANCD2 in DNA Repair

As this thesis primarily focuses on FANCD2, and mutations that affect FANCD2 and FANCI

monoubiquitination or expression are involved in 95% of FA patients, I will focus primarily on

the known roles of FANCD2 within DNA repair. With the exception of putative alpha helical

repeats which are postulated to promote protein/protein interactions, FANCD2 does not contain

any other conserved domains which hint a biological function, and attempts to generate a

FANCD2 crystal structure have been unsuccessful to date (Nookala et al, 2007). While it

remains possible that FANCD2 acts as a molecular scaffold, no DNA repair or replication factor

25

has been identified which requires FANCD2 expression for its localization to DNA damage

induced nuclear foci. Initial reports of a role for FANCD2 in promoting RAD51 focus formation

after DNA damage have been disputed in subsequent studies (Digweed et al, 2002; Wang et al,

2004b; Godthelp et al, 2002; Godthelp et al, 2006).

The cellular hypersensitivity of FA cells to DNA interstrand crosslinks (ICLs) strongly suggests

a role for FA pathway proteins in crosslink repair. Following ICL induction, FANCD2 is

monoubiquitinated and accumulates in nuclear foci with other DNA repair factors including

NBS1, BRCA2, and ATR (Nakanishi et al, 2002; Wang et al, 2004b; Andreassen et al, 2004).

FA cells exhibit increased chromosome breakage and radial formation after ICL treatment, and

cells accumulate with 4N DNA content. Both the chromosome breakage/radial phenotype and

accumulation of FA cells with a 4N DNA content require progression through S phase,

suggesting a role for the FA pathway in repair of ICLs during replication (Akkari et al, 2001).

Analysis of ICL induced radials in FA core complex deficient cells, as well as in complemented

and wild type cells treated with high doses of ICL inducing agents, shows that radials almost

always form between nonhomologous chromosomes (369 out of 372 radials) and the few radials

involving homologs occurred between distant parts of the chromosomes (Newell et al, 2004).

Whether radials form between short regions of homology or non-homologous sequences is

presently unknown, as is whether radials represent an aberrant, or a normal intermediate

structure that has not been properly resolved. The increase in radials in FA cells may reflect an

increase in recombination events using short regions of homology that would normally be

limited, or a problem with resolution of structures. Alternately, radials may form do to an

increase in an end-joining type process which occurs more frequently when the FA pathway is

not present, or may be a reflection of an increase in breaks.

Studies in the Xenopus egg extract system of the repair of a substrate with a single ICL support a

direct role for FANCD2 in replication coupled ICL repair. The initial stages of ICL repair in this

system involve transient stalling of dual replication forks ∼20-40 nucleotides (nt) from the

lesion, then a single fork approaches the lesion and stalls 1 nt from the ICL (Räschle et al, 2008).

This is followed by strand incision on both sides of the ICL on the parental strand, addition of a

nucleotide across from damaged base on the nascent strand, followed by extension beyond the

damaged base by the translesion polymerase ζ. Final repair of both DNA duplexes likely

involves incision repair to remove the damaged base and homologous recombination. Depletion

26

of FANCD2 from Xenopus extracts results in a ∼14 fold decrease in the efficiency of ICL repair,

with significant inhibition of both the incision and extension steps in this process (Knipsheer et

al, 2009). Over time there is an increase in extension products, but this does not result in a

proportional increase in perfect repair products, suggesting that either the extension product

contains errors, or there are additional problems with excision of the damaged base or

recombinational repair. The function of FANCD2 within ICL repair is dependent on its

monoubiquitination, and cannot be complemented by addition of wild-type FANCI in

conjunction with a nonmonoubiquitinatable form of FANCD2.

Insertion of a nucleotide across from a modified base requires utilization of a unique group of

polymerases, in a process known as translesion synthesis. Translesion synthesis is an error prone

process and can result in the introduction of point mutations. Genetic evidence, as well as

analysis of DNA damage induced mutagenesis suggests a role for the FA pathway in this

process, although all members of the FA pathway may not play equivalent roles in promoting

translesion synthesis (Thompson and Hinz, 2009). A recent study examining mutation

frequencies in a plasmid based system, shows that human FA core complex deficient cells have

reduced spontaneous and UVC induced mutation rates relative to complemented controls, but

FANCD2 and FANCI deficient cells have normal mutation rates (Mirchandani et al, 2008). The

hypomutability phenotype in core complex deficient cells may be related to a decrease in foci

formation of the Rev1 translesion polymerase in FANCA and FANCG mutant cells

(Mirchandani et al, 2008). FA core complex proteins may primarily be involved in promoting

translesion synthesis steps in ICL repair, while FANCD1, D2, I, and N may have additional roles

in the final steps of repair involving homologous recombination.

Multiple lines of evidence suggest a role for FANCD2 within homologous recombination

pathways, but exactly what that role is remains unclear. Early studies on primary FA fibroblasts

show increased in vitro interplasmid homologous recombination using FA cell lysates (∼10-20

fold greater then controls) and in vivo intra-plasmid recombination in FA cells (∼50-100 fold

greater then controls) (Thyagarajan and Campbell, 1997). The FA complementation groups are

not stated within this study, but likely correspond to FA core complex components. Subsequent

experiments performed on SV40 immortalized FANCA, FANCG, and FANCD2 cells show an

∼2 fold decrease in efficiency of homology directed repair of an induced I-Sce1 break integrated

27

into the genome in FA deficient cells relative to complemented counterparts (Nakanishi et al,

2005). Possible factors contributing to the different results in these studies include the primary vs

SV40 immortalized status of the cells, the use of cells from a wild-type donor vs complemented

cells as a control, and the use of an extra-chromosomal vs integrated substrates. Importantly, the

study by Tyagarajan and Campbell monitored spontaneous recombination, whereas the study by

Nakanishi and colleagues examined repair of an induced double-strand break. FANCD2 may

have roles both in suppressing certain types of spontaneous recombination events, and in

promoting homology directed DNA repair of an induced break.

Further evidence for a role of FANCD2 in regulating recombination comes from studying

meiotic recombination in FA deficient mouse spermatocytes. FANCA and FANCD2 deficient

spermatocytes have an increased frequency in both mispaired and unpaired chromosomes

(Houghtaling et al, 2002; Wong et al, 2003). FANCD2 also localizes to recombinational nodules

during meiosis in mice, arguing that chromosome pairing abnormalities are a direct consequence

of lack of FANCD2 (S. Meyn, unpubl.).

Additional evidence that FANCD2 may be involved in homologous recombination is indirect,

relying primarily on studies looking at the localization of FANCD2 and its protein interactions.

Following photoinduction of DNA damage, FANCD2 shows tight spatial colocalization with

RAD51, the major human recombinase, at sites of induced damage at a time when

recombinational repair is likely to be ongoing (P. Bradshaw and S. Meyn, unpubl.).

Colocalization with FANCD2 in nuclear foci with proteins implicated in recombination

including BRCA1, BRCA2, RAD51, and MRE11 have also been observed following treatment

of cells with ionizing radiation (Garcia-Higuera et al, 2001; Wang et al, 2004b; Nakanishi et al,

2002). Yeast two-hybrid studies suggest that FANCD2 can directly interact with BRCA2, and

FANCD2 coimmunoprecipitates with BRCA2 in untreated cells and cells exposed to ICLs

(Hussain et al, 2004; Wang et al, 2004b). The interaction between FANCD2 and BRCA2 does

not require FANCD2 ubiquitination but is dependent on FANCG expression, which also

interacts with BRCA2 (Hussain et al, 2003; Wilson et al, 2008). Coimmunoprecipitation

experiments suggest the existence of complex minimally composed of FANCD2, BRCA2,

FANCG, and the RAD51 paralog XRCC3 (Wilson et al, 2008). While this complex has been

postulated to function within recombination, to date it remains unclear what role it plays.

28

FANCD2 also colocalizes and coimmunoprecipitates with BLM, the helicase implicated in

Bloom syndrome, following cellular treatment with interstrand crosslinking and replication fork

stalling agents (Pichierri et al., 2004). Bloom syndrome cells display elevated levels of sister

chromatid exchanges and other chromosomal abnormalities including increased chromatid

breaks, gaps, radials, telomere associations, anaphase bridge and lagging chromosomes

(Chaganti et al, 1974; German and Crippa, 1996, Lillard-Wetherell et al, 2004). The increase in

sister chromatid exchanges in Bloom syndrome has been tied to a role for BLM in dissolution of

double Holliday junction structures (Wu and Hickson, 2003). Additional background on the

relationship between FANCD2 and BLM is provided in the introduction of chapter 3.

One major proposed function of homologous recombination in human cells is to help deal with

replication forks that have encountered lesions and have become stalled or collapsed, which can

result in production of DNA gaps or one-sided double-strand breaks if unrepaired (Michel et al,

1997). Potential mechanisms of both recombination dependent and independent pathways of

dealing with stalled and or broken replication forks are reviewed in Li and Heyer, 2008. Certain

sequences within the genome are more prone to experience replication fork stalling or breakage,

and are referred to fragile sites. Fragile sites share common characteristics including frequent

gaps or breaks when cultured under conditions of replicative stress, such as growth in the

presence of DNA polymerase inhibitors (Glover, 1984). Additionally, fragile sites are frequently

involved in sister chromatid exchanges, translocations, viral integrations, and are often

rearranged or deleted in tumor cells (Howlett et al, 2005).

In vitro experiments show that telomeric repeats are difficult to replicate, and show evidence of

frequent replication fork regression, a mechanism following replication fork stalling that

involves a template switch and lesion bypass without recombination (Fouché et al, 2006). Recent

in vivo experiments further suggest that mammalian telomeres may resemble fragile sites, as they

show increased levels of an abnormal staining pattern with FISH when cells are grown in the

presence of polymerase inhibitors (Sfeir et al, 2009). However other fragile site features such as

increased sister chromatid exchanges are normally suppressed, although may be elevated in

telomere proximal regions (Sfeir et al, 2009). While normal mammalian telomeres share a partial

resemblance with fragile sites, telomeres in ALT cells naturally meet all of the fragile site

criteria, even without the addition of exogenous replication stress. ALT telomeres naturally

contain single-strand gapped regions, show evidence of frequent rearrangements, are prone to

29

undergo sister chromatid exchanges, and frequently integrate DNA in telomere proximal regions

(Nabetani and Ishikawa, 2009; Varley el al, 2002; Londoño-Vallejo et al, 2004; Jegou et al,

2009).

Replication fork stalling agents rapidly induce both FANCD2 monoubiquitination and foci

formation (Hussain et al, 2004; Howlett et al, 2005). Immunofluorescent analysis shows high

levels of FANCD2 colocalization with both RAD51 and PCNA, 90 minutes post treatment of

cells with hydroxyurea to stall forks, suggesting that FANCD2 is involved in the recombinational

response to stalled or collapsed forks (Hussain et al, 2004). When FANCD2 is depleted and cells

are grown in the presence of polymerase inhibitors, there is a 3–4 fold increase in the overall

frequency of chromosome breaks and gaps, and a 2-3 fold increase in gaps and breaks at the

FRA3B and FRA16D fragile sites relative to controls (Howlett et al, 2005). This suggests that

FANCD2 normally suppresses genomic instability at fragile sites, as well as other loci within the

genome. Confirming a role for FANCD2 at fragile sites, replication stress induces high levels of

mitotic cells containing sister chromatids with paired FANCD2 foci that localize to common

fragile sites (Chan et al, 2009). Approximately 10% of FANCD2 foci are connected by ultra-fine

bridges coated with BLM during anaphase, with bridges likely representing unresolved

replication intermediates. The number of FANCD2 foci exceeds the number of breaks at fragile

sites, suggesting that FANCD2 not only responds to broken fragile sites, but also intact fragile

sites containing abnormal constrictions or linkages (Chan et al, 2009).

1.3 Concluding Remarks

As telomere dysfunction is one potential cause of bone marrow failure, and telomere

abnormalities have been reported in cells from FA patients, the goal of this project was to

elucidate what role, if any, the FA pathway plays within telomere maintenance. Experiments

focused primarily on FANCD2, because the majority of known patient mutations directly affect

FANCD2 function, and the relationship between FA core complex proteins and more

downstream FA proteins (FANCD1/BRCA2, FANCN/PALB2, FANCJ/BACH1) is presently

unclear. Given the known and proposed functions of FANCD2, there are multiple ways in which

FANCD2 may impact telomere maintenance including promoting t-loop formation, limiting

telomere rapid deletion events, aiding in telomeric replication, and preventing telomeres from

expressing characteristics of fragile sites. While I did not obtain results supporting a role for

30

FANCD2 in maintenance of telomeres in a setting where telomerase is expressed, I did find

significant roles for FANCD2 within the ALT telomere maintenance pathway. Through the

further exploration of the role of FANCD2 within ALT, I have begun to understand more about

functions of FANCD2 within recombination, and the relationships between FANCD2 and DNA

repair factors it associates with.

31

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Zou Y, Sfeir A, Gryaznov SM, Shay JW, Wright WE (2004). Does a sentinel or a subset of short telomeres determine replicative senescence? Mol Biol Cell. 15:3709-18.

Zou Y, Misri S, Shay JW, Pandita TK, Wright WE (2009). Altered states of telomere deprotection and the two-stage mechanism of replicative aging. Mol Cell Biol. 29:2390-7.

Zschenker O, Kulkarni A, Miller D, Reynolds GE, Granger-Locatelli M, Pottier G, Sabatier L, Murnane JP (2009). Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line. DNA Repair. 8:886-900.

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Chapter 2

2 The Fanconi Anaemia Pathway Plays a Critical Role in Cells that Utilize the Alternative Pathway of Telomere Maintenance

2.1 Abstract

Fanconi anaemia (FA) is a multigenic pleiotrophic syndrome characterized by bone marrow

failure, high cancer rates and congenital malformations. FA gene products have been implicated

in homologous recombination, a process involved in telomere maintenance. Here, I show that

FANCD2, A, and G localize to telomeric foci in human cells that utilize the recombination based

Alternative Lengthening of Telomeres (ALT) pathway, but not telomerase positive cells.

FANCD2 localizes to those telomeric foci that contain BLM, and coimmunoprecipitation

experiments indicate interactions between FANCD2, BLM, and TRF2 in late S/G2 ALT cells.

Interestingly, localization of FANCD2 to telomeric foci requires BLM expression. FANCD2

typically localizes to telomeric foci that have not activated a DNA damage response, and

FANCD2 localization to telomeric foci is largely independent of ATM and TRF2, but requires

monoubiquitination by the FA core complex. Only ∼20% of FANCD2 colocalization events with

telomeric foci are dependent on ATR expression, arguing that the primary function of FANCD2

is not replication fork rescue/restart.

FANCD2 primarily colocalizes with telomeric proteins within ALT-associated PML Bodies

(APBs). Electron spectroscopic imaging of APBs reveals that the bodies are composed of a

nucleic acid and protein interior, surrounded by a outer protein layer. The morphology and

phosphorous signal intensity of the nucleic acid within APBs differs from chromatin, strongly

suggesting that this material does not represent telomeres themselves, but rather extra-

chromosomal telomeric material. When FANCD2 is depleted there is an ALT-specific increase

in telomere dysfunction induced foci, and APBs are occasionally observed with chromatin-like

masses invading the bodies, suggesting that dysfunctional telomeres may preferentially associate

with APBs. ALT cells depleted of FANCD2 have reduced viability and show signs of mitotic

catastrophe: supernumerary centrosomes, rereplicated DNA, and aneuploidy. Together my

50

results suggest that FANCD2 has an essential BLM-dependent function in ALT cells that may be

independent from its role in the response to DNA damage and replication fork rescue.

2.2 Introduction

Homologous recombination plays important roles in DNA repair, replication and telomere

maintenance. Fanconi anaemia (FA) is rare genetic syndrome clinically characterized by

progressive bone marrow failure, increased cancer incidence and congenital abnormalities

(Kutler et al, 2003; Auerbach AD, 2009). FA cells exhibit hypersensitivity to DNA crosslinking

agents, DNA repair defects, and spontaneous chromosomal aberrations (Weksberg et al, 1979;

Schroeder and Kurth, 1971). Seven FA proteins (FANCA, B, C, E, F, G, L) participate in a core

complex that is required to promote monoubiquitination and foci formation of FANCD2 and

FANCI during replication and after DNA damage (Garcia-Higuera et al, 2001; Medhurst et al,

2006; Smogorzewska et al, 2007). Additional repair proteins, including FANCM, ATM, ATR,

RPA, HCLK2, RAD17, RAD9, CHK1 and BRCA1, are involved in promoting efficient

FANCD2 monoubiquitination after cellular exposure to exogenous DNA damaging agents

(Bakker et al, 2009; Ho et al, 2006; Andreassen et al, 2004; Collis et al, 2007; Guervilly et al,

2008; Garcia-Higuera et al, 2001). Evidence to date suggests that the FA pathway plays roles in

the response to stalled/collapsed replication forks, crosslink repair, and in recombination.

Supporting a role for the pathway in recombination, BRCA2 and its binding partner PALB2 are

mutated in the FANCD1 and FANCN complementation groups, (Howlett et al, 2002; Reid et al,

2007) and reporter assays show impaired recombinational repair in FA core complex and

FANCD2 mutant cells (Nakanishi et al, 2005). FANCD2 also colocalizes with RAD51 at sites of

photo-induced DNA breaks and in recombination nodules during meiosis (Bradshaw and Meyn,

unpubl.), and FANCA and FANCD2 knockout mice show increased incidence of unsynapsed

axial elements and chromosome mispairing during meiosis (Wong et al, 2003; Houghtaling et al,

2003).

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes, preventing ends

from activating cell cycle checkpoints or becoming substrates for DNA repair reactions that

could result in telomere fusions or rapid shortening events. Proper capping of chromosome ends

depends not only on sufficient telomere length and the presence of telomere binding proteins, but

also on DNA repair factors implicated in global repair pathways (Longhese MP, 2008). DNA

51

repair factors may be involved in the processing of chromosome ends, as well as formation and

maintenance of the t-loop, a large duplex loop that is formed when the 3’ssDNA telomeric

overhang invades its own proximal dsDNA telomeric DNA (Griffith et al, 1999). Additional

DNA repair factors are required to promote replication of telomeres, as the repetitive nature and

propensity of the TTAGGG sequence to form secondary structures can result in frequent

replication fork stalling (Gilson and Géli, 2007).

Telomeres in cycling somatic cells typically shorten with age, eventually becoming uncapped

and involved in aberrant end-joining reactions, leading to cellular senescence or apoptosis (Zou

et al, 2004). The few cells that bypass this fate enter a period of widespread genomic instability

and cellular crisis, with rare survivors emerging that have activated or up-regulated a mechanism

of telomere maintenance (Kim et al, 1994; Bryan et al, 1995; Bryan et al, 1997). Approximately

90% of immortalized cells rely on telomerase to add de novo repeats to telomeres, while the

remaining cells depend on the Alternative Lengthening of Telomeres (ALT) pathway(s) (Bryan

et al, 1997). The mechanism of ALT is still being elucidated, but evidence to date supports a role

for both inter- and intra-telomeric recombination (Dunham et al, 2000; Londoño-Vallejo et al,

2004; Muntoni et al, 2009). ALT cells also typically contain high amounts of linear and circular

single- and double-strand Extra-Chromosomal Telomeric Repeat (ECTR) DNA that may be a

product, as well as a substrate in recombination reactions (Cesare AJ and Griffith JD, 2004;

Nabetani A and Ishikawa F, 2009).

Evidence of excessive telomere shortening relative to age-matched controls has been reported in

peripheral blood samples in FA patients, and correlates with progression towards aplastic

anaemia (Leteurtre et al, 1999; Hanson et al, 2001; Callen et al, 2002). However, a recent survey

found that telomere shortening is not equal among all haematopoietic lineages, and many FA

patients have average telomere lengths that are in the range of healthy controls (Alter et al 2007).

Average telomere length measurements do not address the frequency of critically short

telomeres, which appear to be the driving force in activation of the DNA damage response at

telomeres, telomere fusions, and induction of cellular senescence (Zou et al, 2004). Techniques

measuring individual telomere lengths have also resulted in conflicting results, with one group

showing no difference in telomere length distribution between FA patients and controls (Franco

et al, 2004) and another group showing an increased frequency of ends without detectable FISH

signals and excess ECTR DNA (Callen et al, 2002). Telomere abnormalities in FA cells do not

52

appear to be due to problems with telomerase, as telomerase activity appears normal in FA

haematopoietic cells (Leteurtre et al, 1999), and mutations in telomerase have not been identified

(Calado et al, 2004).

The aim of the present study was to clarify the role of the FA pathway in telomere maintenance

and determine whether observed telomere abnormalities are solely a consequence of increased

cell turnover, or are indicative of a telomere maintenance abnormality. My results do not support

a critical role for the FA pathway in telomere maintenance in cells that rely on telomerase for

telomere maintenance, but indicate an essential role for the pathway in cells that utilize ALT

telomere maintenance.

2.3 Materials and Methods

Cell culturing. GM00847 (GM847), Wi38-VA13/2RA (VA13), U2OS, GM00639 (GM639),

HT1080, GM00637 (GM637), SAOS-2 and PD20 cells were grown in DMEM supplemented

with 10% fetal bovine serum and penicillin-streptomycin. Cells were mycoplasma free. When

required, cells were manually counted using a haemocytometer. When indicated, hydroxyurea or

KU55933 was added prior to cells prior to analysis at concentrations of 2mM for 24 hours, or

10uM for 36 hours, respectively. To verify the stability of KU55933 over a 36 hr period, cells

were incubated with 10uM KU55933 for 36 hours, exposed to 5 Gy of irradiation, and then

analyzed for p53 phosphorylation after 1 hour.

Immunofluorescence microscopy. Cells were grown on glass coverslips, and then processed for

immunostaining as previously described (Stavropoulos et al., 2002). Primary antibodies used

were from Novus Biologicals: rabbit anti FANCD2 (NB100-182), rabbit anti 53BP1 (NB 100-

304); from Santa Cruz Biotechnology: mouse anti-FANCD2 (sc-20022 ), goat anti-TRF1 (sc-

6165), goat anti-FANCA (sc-18664), goat anti-BLM (sc-7789); from Imgenex: mouse anti-TRF2

(IMG-124); and from Millipore: rabbit anti-PML (AB1370). Rabbit anti-FANCA and rabbit anti-

FANCG were a kind gift from Dr. Manual Buchwald. Mouse anti-PML 5E10 was a kind gift

from Dr. Roel van Driel. All antibodies were validated for use in IF by examining staining in

cells with reduced protein levels (patient cell lines or cells targeted with siRNA), or by verifying

that multiple independent antibodies (not listed) recognized the same nuclear structures using IF.

53

Images were obtained using a 1.4NA objective mounted onto a Zeiss Axioplan 2 microscope

equipped with a Hammamatsu Orca ER camera. Unless otherwise indicated, images were taken

under 63x magnification. 12-bit grayscale images were captured using Openlab software

(Improvision). Slides from a single experiment were all processed and imaged at the same time,

using identical exposure times. Average nucleoplasmic fluorescence was determined using

Openlab, then images were thresholded so only foci with a 2 fold intensity over background

nucleoplasmic staining were analyzed. Foci number and co-localization were manually

determined by analysis of Openlab images.

Immunoblotting. Cells were lysed in RIPA buffer (150mM NaCl, 10mM Tris pH 7.2, 5mM

EDTA, 0.1% SDS, 1.0% Triton X-100, 1.0% Na-deoxycholate, protease and phosphatase

inhibitors) and 10-20 ug of lysate was run out on NuPAGE 4-12% Bis-Tris gels (Invitrogen) or

7.5% SDS-PAGE gels. Blots were probed with mouse anti-FANCD2 (sc-20022 Santa Cruz),

mouse anti B-tubulin (sc-5274 Santa Cruz), mouse anti-TRF2 (IMG-124 Imgenex), mouse anti-

ATR (2B5 GeneTex), mouse anti p53 (Ab-6 Oncogene), rabbit anti-BLM (NB100-214 Novus

Biologicals), rabbit anti-p53 (pSer15) (PC386 Calbiochem). Secondary antibodies were labeled

with HRP (Jackson ImmunoResearch) and chemiluminescent detection using ECL was carried

following the manufacturers’ instructions (GE Healthcare).

Immunoprecipitation. VA13 and GM639 cells were synchronized using a double thymidine

block and harvested 8-9 hr post release when cells had accumulated in late S-G2.

Synchronization was verified by analysis of DNA content, and immunofluorescent analysis of

cell cycle markers. Cells were rinsed twice with PBS containing protease inhibitors, harvested on

ice via mechanical detachment, incubated in lysis buffer for 15 min (150mM NaCl, 50mM Tris

pH 7.4, 0.5% NP40, 1x protease inhibitor cocktail (Roche), 0.5mM PMSF, 50mM NaF, 1mM

NaOrthovanadate, 38mM 4-Nitrophenyl phosphate disodium salt, 1mM β-glycerophosphate) and

sheared 5x with 18, 23 and 25 gauge needles. Cellular debris were pelleted (10 000g x 10min at

4°C), protein concentration was determined, 3.5 mg of protein was aliquoted into eppendorf

tubes, and volumes were equalized between cell lines with lysis buffer. Lysates were precleared

overnight at 4°C with protein G sepharose beads, then incubated with 2.5 µg rabbit anti-TRF2

(SC-9143 Santa Cruz), rabbit anti-FANCD2 (NB100-182 Novus Biologicals), rabbit anti-BLM

(NB100-161 Novus Biologicals), rabbit anti-GAL4 (SC-577 Santa Cruz) at 4°C, and incubated

with protein G sepharose beads for 3 hrs at 4°C. Beads were washed 3 x 15 min in 150mM NaCl,

54

50mM Tris, 0.2% NP40, resuspended in 15ul SDS PAGE loading buffer, boiled for 10 min, then

protein was run on 7.5% denaturing acrylamide gels and western blotted.

siRNA. siRNA oligonucleotides were synthesized (Dharmacon) to target the following

sequences: FANCD2 (A, 5’-GGAGATTGATGGTCTACTA-3’ Zhu and Dutta 2006; B, 5’-

CCAGGAAGCAACCACTTTC-3’; C, FANCD2 siRNA (h): sc-35356 from Santa Cruz; D, 5’-

AACAGCCATGGATACACTTGA-3’ Howlett et al., 2005; Fan et al., 2009) ATR (5’-

AACCTCCGTGATGTTGCTTGA-3’ Andreassen et al., 2004) FANCA (5’-

AAGGGTCAAGAGGGAAAAATA-3’ Bruun et al., 2003; Andreassen et al., 2004) BLM (5’-

GAGCACATCTGTAAATTAA-3’) TRF2 (5’-GAAGTGGACTGTAGAAGAA-3’) and control

GL2 (5’-AACGTACGCGGAATACTTCGA-3’ Zhu and Dutta 2006). 1 x 105 – 2 x 105 cells in

a single well of a 6 well plate were transfected with 50-100 nM siRNA using Lipofectamine

RNAiMax (Invitrogen) following manufacturers instructions. Cells were subjected to a second

round of siRNA, 48 hours after the first transfection and analyzed 5 days after the initial

transfection unless otherwise indicated.

Transfections. Transfections of FANCD2-GFP constructs were performed using Fugene 6

(Roche) following manufacturers instructions. pMMP-puro-EGFP-FANCD2 was a generous gift

from Dr. Alan D’Andrea. The Quick change II XL site directed mutagenesis kit was used to

introduce a point mutation in pMMP-puro-EGFP-FANCD2 changing FANCD2 lysine 561 to

arginine.

ImmunoFISH. Cells were fixed and immunostained as described above. Following the

immunostaining, cells were fixed in 4% paraformaldehyde in PBS for 20 min, than FISH was

performed as described (Eller et al., 2006). Telomeric DNA was detected with a 0.5ug/ml

telomere PNA probe (Rho-(C3TA2)3).

FISH. Cells for FISH experiments were harvested, subjected to hypotonic swelling in 75mM

KCl (15min at 37°C), fixed in methanol/acetic acid, and dropped onto slide following standard

protocols. For analysis of signal free ends, cells were incubated in 0.1ug/ml colcemid for 1-2

hours prior to harvesting. FISH was carried out as previously described (Zijlmans et al., 1997)

with minor modifications. Hybridization mixture containing 70% formamide, 0.5ug/ml telomere

PNA probe (Rho-(C3TA2)3), 0.5ug/ml FITC-pan-centromeric PNA probe (Tabori et al, 2006),

10mM Tris pH 7.2, 0.1% blocking reagent (Boehringer), MgCl2 buffer (4.1mM Na2HPO4,

55

0.45mM citric acid, 1mM MgCl2) was preheated for 3 min at 86°C, added to slides, covered with

a coverslip, than slides were heated for 3 min at 81°C and left for 2 hours at room temperature

prior to washing. Slides were washed 2 x 15 min in 70% formamide, 10mM Tris pH7.2, 0.1%

BSA, and then 3 x 5 min in 100mM Tris pH 7.2, 150mM NaCl, 0.08% Tween 20.

Electron spectroscopic imaging. Experiments were carried out by Andrew Larsen with

technical assistance from Dr. Ren Li. For a detailed explanation of methods used please refer to

the thesis by Andrew Larsen to be submitted to the Department of Biochemistry, University of

Toronto, 2010.

Colony forming assays. Cells transfected with FANCD2 siRNA A or GL2 control siRNA were

replated 24 hours after the second siRNA transfection into 6 cm plates at densities of 125, 250,

500, 1000, and 10 000 cells/plate. All cells were plated in duplicate and experiments were

repeated 4 times. When clearly visible colonies appeared (8-16 days post plating) plates were

rinsed in PBS, fixed in methanol for 15 minutes, stained in 10% Giemsa for 15 minutes, rinsed in

ddH2O, dried, and manually counted.

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2.4 Results

2.4.1 FANCD2 localize to telomeric foci and PML bodies in ALT human cells

FANCD2 forms nuclear foci during S phase and in response to induced DNA damage (Garcia-

Higuera et al, 2001). I find that in cells that utilize the ALT pathway of telomere maintenance,

FANCD2 forms nuclear foci that colocalize with telomere binding proteins (TRF1/TRF2) and

telomeric DNA (Figure 2-1A). TRF1 and TRF2 are major components of the shelterin telomere

binding complex (de Lange, 2005), and are commonly used as markers of telomeric DNA.

Localization of FANCD2 to telomeric foci in ALT cells was verified with multiple independent

FANCD2 and TRF1 and TRF2 antibodies, and is in accord with recent studies (Spardy et al,

2008; Fan et al, 2009). FANCD2 colocalization with telomeric foci was rarely observed in

telomerase positive cell lines (Figure 2-1 A, B) or primary cells (data not shown) and never

exceeded more then one or two foci per nucleus. Whether FANCD2 localizes to replicating or

dysfunctional telomeres in telomerase positive cells is currently unknown, however the fact that

occasional colocalization events are detected argues against the idea that FANCD2 is present at

most telomeres, but is undetectable by immunofluorescence. Within ALT cells there is a cell

cycle-dependent variability in the extent of FANCD2 colocalization. Similar to Fan et al, I find

that colocalization between FANCD2 and TRF2 is maximal in cells during late S/G2 (data not

shown), with lower levels observed in all other phases of the cell cycle.

ALT associated PML Bodies (APBs) are a subtype of PML nuclear body that is characterized by

the association of telomeric DNA sequence and binding proteins with normal PML body

components (Yeager et al, 1999). The function of APBs is currently unknown, however they are

most common during late S/G2 when recombination is known to occur, suggesting that they are

potential sites of recombination reactions between telomeres. Colocalization analysis shows that

FANCD2 localizes to APBs (Figure 2-2 A), and that ∼80% FANCD2 colocalization with

telomeric proteins occurs at APBs (Figure 2-2 B). FANCD2 only rarely colocalizes with non-

telomeric PML bodies in ALT and telomerase positive cells (Figure 2-2 B), the significance of

which is unknown.

57

A) DAPI FANCD2 Telomeric DNA TRF2 Merge

B)

Figure 2-1. FANCD2 frequently colocalizes with telomeric DNA and telomere binding

proteins in cells that utilize the ALT pathway, but not in telomerase positive cells A) Representative images of GM847 and VA13 ALT nuclei with a high degree of colocalization between

FANCD2 and telomeric DNA and binding proteins, and HT0180 and GM639 telomerase positive nuclei

where FANCD2 does not frequently colocalize with telomeric DNA or binding proteins. Scale bar is

5µm. B) Plot of the number of FANCD2 foci that do, and do not colocalize with TRF2 from 300

randomly selected asynchronous cells from 3 independent experiments ± the associated standard error for

each cell line.

GM847 (ALT)

VA13 (ALT)

HT1080 (telomerase

positive)

GM639 (telomerase

positive)

FANCD2 foci not colocalized with TRF2 FANCD2 foci colocalized with TRF2

58

A) DAPI FANCD2 TRF1 PML

B) GM847 VA13

Figure 2-2. FANCD2 primarily colocalizes with telomeric proteins within ALT-associated

PML Bodies (APBs) A) Representative images of ALT cell nuclei with a high degree of colocalization between FANCD2 and

TRF1. Most FANCD2 foci which colocalize with TRF1 also colocalize with PML, and therefore

represent APBs. Scale bar is 5µm. B) Venn plots showing colocalization between FANCD2, TRF1, and

PML foci from 150 randomly selected asynchronous GM847 (left) or VA13 (right) ALT nuclei selected

over 3 independent experiments.

GM847 (ALT)

VA13 (ALT)

59

2.4.2 FA core complex components localize to ALT telomeric foci and promote FANCD2 monoubiquitination and localization to telomeric foci

In addition to FANCD2, I find that FA core complex components FANCA and FANCG also

colocalize with FANCD2 and TRF1 within ALT cells (Figure 2-3 A). Colocalization analysis on

randomly selected asynchronous ALT cells, shows that 69% and 77% of FANCD2 foci that

colocalize with TRF1 also contain FANCA in GM847 and VA13 cells respectively (Figure 2-3

B). FANCA localization to telomeric foci was confirmed with two independent antibodies, and

signal specificity was confirmed with siRNA knockdown of FANCA. FANCA and FANCG foci

did not colocalize with telomeric proteins in telomerase positive cells (data not shown).

Using siRNA to target FANCA, I was able to significantly reduce both FANCA protein levels

and FANCD2 monoubiquitination (Figure 2-4 A). Analysis of FANCD2 foci formation in

FANCA-depleted cells versus random siRNA treated controls, shows that FANCA knockdown

results in 11 and 15 fold reductions in the number of non-telomeric associated FANCD2 foci in

GM847 and VA13 depleted cells, respectively (Figure 2-4 B). The number of FANCD2 foci

associated with telomeric proteins showed a more modest decrease of 2 and 1.6 fold in FANCA-

depleted GM847 and VA13 cells relative to controls, however residual FANCD2 foci in

FANCA-depleted cells were significantly less intense then controls (Figure 2-4 C).

To ensure that monoubiquitination is required for all FANCD2 accumulation at telomeric foci, I

generated a non-monoubiquitinatable GFP tagged FANCD2 mutant through site-directed

mutagenesis of lysine 561 (GFP-FANCD2K561R) and transiently expressed it in GM847 cells

(Figure 2-4 D). The GFP-FANCD2 wild type construct was generated in the lab of Dr. Alan

D’Andrea, and shown to behave similarly to wild-type FANCD2 (Chirnomas et al, 2006). I find

that in contrast to GFP-FANCD2, which forms foci in 29% of transiently transfected GM847

cells (500 cells analyzed), GFP-FANCD2(K561R) fails to accumulate in visible foci (2000 cells

analyzed). Together these results support a requirement for FANCD2 monoubiquitination at

lysine 561 by the FA core complex for FANCD2 accumulation at ALT telomeric foci.

60

A) DAPI FANCA FANCD2 TRF1

B)

Figure 2-3 FA core complex proteins FANCA and FANCG colocalize with FANCD2 at

ALT telomeric foci A) Representative images of GM847 and VA13 ALT cells with a high degree of colocalization between

FANCA, FANCD2 and TRF1 (upper 2 rows) and FANCG, FANCD2 and TRF1 (lower 2 rows). Scale bar

is 5 µm. B) Venn plots showing colocalization between FANCD2, FANCA, and TRF1 foci from 150

randomly selected asynchronous ALT nuclei selected over 3 independent experiments.

GM847 (ALT)

VA13 (ALT)

GM847 (ALT)

VA13 (ALT)

DAPI FANCG FANCD2 TRF1

GM847 VA13

61

A)

B)

C)

62

D)

Figure 2-4. FANCD2 localization to ALT telomeric foci in GM847 and VA13 ALT cells is

dependent on monoubiquitination by the FA core complex

A) SiRNA depletion of FANCA results in a significant reductions in FANCA protein level and FANCD2

monoubiquitination B) Plot of the number of FANCD2 foci that do, and do not colocalize with TRF2

from 300 randomly selected asynchronous cells from 3 independent experiments ± the associated

standard error. C) Remaining FANCD2 foci in FANCA siRNA treated cells are smaller and less intense

than controls. Scale bar is 10 µm. D) FANCD2-GFP forms foci, while non-monoubiquitinatable

FANCD2 (FANCD2K561R) does not. Scale bar is 10 µm.

2.4.3 FANCD2 localizes to ALT telomeric foci that have not activated a DNA damage response, and localization to telomeric foci is independent of ATM and ATR kinase activity

Telomeres that have become dysfunctional through excessive shortening or disturbance of the

normal capping structure activate a DNA damage response, commonly characterized by the

colocalization of telomere binding proteins with 53BP1 or serine 139 phosphorylated histone

H2AX (γH2AX) (Takai et al, 2003). I monitored colocalization between FANCD2, TRF1, and

53BP1 to determine whether FANCD2 specifically localizes to telomeric foci that have activated

a DNA damage response. I find that in GM847 and VA13 ALT cells, only 15% and 12% of

telomeric foci that FANCD2 localizes to, also contain 53BP1 (Figure 2-5 A), suggesting that

telomeric DNA-associated FANCD2 is not simply participating in the response to dysfunctional

telomeres.

ATR and ATM are PI-3-related kinases that play key early signaling roles in the response to

DNA damage. ATR is primarily involved in coordinating the response to replication stress, and

ATM is involved in the response to double-strand breaks. FANCD2 monoubiquitination and foci

formation are strongly induced by agents that cause replication stress, and ATR appears to play a

63

key role in promoting this process (Andreassen et al, 2004). Replication stress can also stimulate

formation of large APBs that contain FANCD2, and this process appears partially dependent on

ATR (Spardy et al, 2008). I wanted to determine whether ATR is required for FANCD2 to

localize to normal endogenously forming APBs. SiRNA was used to transiently knockdown

ATR in GM847 and VA13 ALT cells. To verify that the level of knockdown obtained was

functional, I treated cells with hydroxyurea, a replication fork-stalling agent, and monitored the

affect of ATR depletion on FANCD2 monoubiquitination. The level of ATR depletion was

sufficient to decrease FANCD2 monoubiquitination in response to hydroxurea in GM847 and

VA13 ALT cells (Figure 2-5 B). However, when FANCD2 foci formation was monitored, I

observed a slight increase in the number of non-telomeric FANCD2 foci, and only a ∼ 20%

decrease in FANCD2 colocalization to telomeric foci (Figure 2-5 D). Residual FANCD2 foci

that formed in ATR depleted cells were of similar intensity to foci in controls (data not shown).

If FANCD2 was primarily responding to stalled/collapsed replication forks present in APBs and

telomeric foci, I would predict that localization of most FANCD2 would be largely dependent on

ATR expression, which was not observed.

ATM also influences FANCD2 monoubiquitination and foci formation after exposure of cells to

DNA damaging agents (Ho et al, 2006), and promotes efficient formation of telomere

dysfunction induced foci (Takai et al, 2003). To determine whether ATM activity is required for

FANCD2 localization, GM847 and VA13 cells were grown in media containing the ATM

inhibitor KU55933, resuspended in DMSO, for 36 hours, then FANCD2 foci formation and

localization were assessed. Inhibition of ATM activity was verified by monitoring suppression of

p53 serine 15 phosphorylation, a known ATM target, after irradiation (Figure 2-5 C). I find that

inhibiting ATM activity with KU55933 has no significant affect on FANCD2 localization to

telomeric foci, and causes only a slight decrease in non-telomeric foci formation foci (Figure 2-5

D). Similar results were observed when cells were grown in 20 or 40 µM wortmannin to inhibit

ATM activity (data not shown). This suggests that FANCD2 is not simply responding to

telomeric DNA structures as part of an ATM coordinated DNA damage response.

64

A) GM847 VA13

B)

C)

65

D)

Figure 2-5. FANCD2 localization to ALT telomeric foci is not simply part of a DNA

damage response A) Venn plots showing colocalization of FANCD2, 53BP1, and TRF1 foci from populations of 150

randomly selected asynchronous GM847 and VA13 ALT cells. FANCD2 primarily localizes to telomeric

foci that have not activated a DNA damage response. B) SiRNA depletion of ATR is sufficient to limit

FANCD2 monoubiquitination in GM847 and VA13 cells treated with 2mM HU for 24 hours. C)

KU55933 inhibition of ATM activity is sufficient to limit p53 phosphorylation in GM847 and VA13 cells

when measured 1 hour post treatment with 5 Gy IR. D) Plot of the number of FANCD2 foci that do, and

do not colocalize with TRF2 from 300 randomly selected asynchronous cells from 3 independent

experiments ± the associated standard error.

2.4.4 FANCD2 coimmunoprecipitates with TRF2 and BLM in ALT cells, and almost always localizes to telomeric foci that also contain BLM

TRF2 appears to be involved in telomeric recombination, as in vitro experiments show that it

promotes t-loop formation and binds to the base of the loop where a partial or full Holliday

junction is present (Griffith et al, 1999). Further in vitro evidence suggests that TRF2 has both

pro and anti-recombinogenic activities, and both promotes formation of Holliday junctions and

prevents their subsequent resolution (Poulet et al, 2009). BLM, the helicase mutated in Bloom

syndrome, also has activities that promote and inhibit recombination, and coimmunoprecipitates

GM847 GM847 VA13 VA13 GM847 GM847 VA13 VA13 +random +ATR +random +ATR +DMSO +KU55933 +DMSO +KU55933 siRNA siRNA siRNA siRNA

66

with TRF2 in ALT cells but not telomerase positive cells (Lillard-Wetherell et al, 2004).

Additionally, fluorescent resonance energy transfer experiments support a direct in vivo

interaction between BLM and TRF2 in ALT cells (Stavropoulos et al, 2002), and in vitro

experiments show that TRF2 stimulates BLM strand unwinding activity (Lillard-Wetherell et al,

2004). FANCD2 has been implicated in recombination, binds to Holliday junctions with high

affinity in vitro (Park et al, 2005), and interacts with BLM after DNA damage (Pichierri et al,

2004). Therefore I performed coimmunoprecipitation experiments testing for in vivo interactions

between FANCD2, TRF2, and BLM. VA13 ALT cells and GM639 telomerase positive cells

were synchronized with a double thymidine block, and harvested 8-9 hrs after release to enrich

for cells in late S/G2, when telomeric recombination is believed to occur.

I find that when FANCD2 is immunoprecipitated, TRF2 and BLM are coimmunoprecipitated in

VA13 ALT cells, but not GM639 telomerase positive cells (Figure 2-6 A). Immunoprecipitation

of TRF2 also results in coimmunoprecipitation of FANCD2 in VA13 cells, but not in GM639

cells (Figure 2-6 B). Only a small amount of protein was coimmunoprecipitated in all cases,

suggesting that interactions may involve a small subset of each protein, or be weak or transient in

nature. Supporting an interaction between FANCD2, BLM and TRF2 in ALT cells, in

asynchronous GM847 and VA13 ALT cells, 90 and 94% of telomeric foci that contain FANCD2

also contain BLM, respectively (Figure 2-6 C). This suggests that FANCD2 may be

participating in a common process with BLM and/or that both proteins respond to the same DNA

substrates.

2.4.5 FANCD2 localization to APBs is independent of TRF2, but requires BLM expression

SiRNA knockdown of TRF2 significantly reduces TRF2 protein levels (Figure 2-7 A) and foci

formation 72 hours after siRNA addition, (Figure 2-7 B) but does not affect TRF1 foci formation

or APB formation (Figure 2-7 B,C), demonstrating that TRF1 and TRF2 associate independently

with telomeric DNA, and that APB formation is not dependent on TRF2, consistent with results

by Stagno D'Alcontres et al. (2007). FANCD2 colocalization with APBs, and with TRF1 foci not

associated with PML, does not require TRF2 expression, but rather increases slightly in TRF2

depleted 847 and VA13 ALT cells (Figure 2-7 C). The decrease in the amount of non-

monoubiquitinated FANCD2 in TRF2 siRNA treated cells may be a result of alterations to the

cell cycle, as siRNA depletion of TRF2 resulted in a high degree of cell death (data not shown).

67

A) B)

C) GM847 VA13

Figure 2-6. FANCD2 interacts with BLM and TRF2 in late S/G2 ALT, but not telomerase

positive cells

A) Immunoprecipitation of FANCD2 in late S/G2 VA13 ALT cells, but not late S/G2 GM639 telomerase

positive cells, coimmunoprecipitates BLM and TRF2. B) Immunoprecipitation of TRF2 in late S/G2

VA13 ALT cells, but not late S/G2 GM639 telomerase positive cells, coimmunoprecipitates FANCD2.

C) Venn plot showing colocalization of FANCD2, BLM, and TRF2 foci in populations of 150 randomly

selected GM847 and VA13 asynchronous cells selected from 3 independent experiments. FANCD2

primarily localizes to ALT telomeric foci that also contain BLM.

68

A)

B)

C)

Figure 2-7 FANCD2 localization to ALT telomeric foci is independent of TRF2 expression A) TRF2 siRNA significantly reduces TRF2 protein levels in GM847 and VA13 ALT cells. B) TRF2

knockdown does not affect TRF1 or FANCD2 foci formation or colocalization in VA13, or GM847 cells

(not shown). C) Venn plots showing colocalization between FANCD2, PML and TRF1 foci in

asynchronous populations of 150 GM847 and VA13 cells treated with random or TRF2 siRNA.

GM847 + random siRNA GM847 + TRF2 siRNA

VA13 + random siRNA VA13 + TRF2 siRNA

69

In contrast to TRF2 knockdown, transient depletion of BLM with siRNA leads to a dramatic

reduction in FANCD2 colocalization with telomeric foci. BLM knockdown does not

significantly alter FANCD2 monoubiquitination (Figure 2-8 A), nor does BLM depletion

decrease the number of and non-telomeric FANCD2 foci (Figure 2-8 B). However, BLM

depletion causes 79% and 84% reductions in the number of FANCD2 foci colocalized with

TRF2 in 847 and VA13 cells, respectively. The fraction of cells in S and G2 does not

significantly differ in BLM and random siRNA treated cells, and APBs continue to form at a

similar frequency, however very large APBs are rarely observed (data not shown). BLM is not

required for FANCD2 S phase foci, and is not required for FANCD2 foci formation after

exposure of cells to exogenous DNA damage (Pichierri et al, 2004), making the BLM

dependency of FANCD2 colocalization to ALT telomeric foci a novel behaviour.

A)

B)

Figure 2-8 FANCD2 localization to ALT telomeric foci is dependent on BLM expression

A) BLM siRNA significantly reduces BLM protein levels in GM847 and VA13 ALT cells. B)

Plot of the number of FANCD2 foci that do, and do not colocalize with TRF2 from 300

randomly selected asynchronous cells from 3 experiments ± the associated standard error.

GM847 GM847 VA13 VA13 + random + BLM + random + BLM siRNA siRNA siRNA siRNA

70

2.4.6 FANCD2 knockdown causes an ALT-specific increase in telomere dysfunction induced foci that is independent of rapid telomere shortening

To investigate the role of FANCD2 in ALT cells I used a siRNA knockdown approach. I tested

four different siRNAs and found three that reduced expression via western blot analysis to below

the residual amount present in the PD20 FANCD2 patient cell line (Figure 2.9 A). With this level

of silencing, FANCD2 foci were no longer visible by IF under 63x magnification using my most

sensitive antibody (Figure 2-9 B). Transfection efficiency as judged by a fluorescently tagged

random siRNA was approximately 100% in all cell lines tested, and FANCD2 siRNA

significantly decreased in FANCD2 expression in all cell lines tested (data not shown).

Figure 2-9. FANCD2 siRNA significantly

reduces FANCD2 protein levels and foci

formation A) Western blot analysis of FANCD2 protein levels in PD20 cells with biallelic FANCD2 mutations, and GM847 cells treated with random or FANCD2 targeting siRNAs. B) Immmunofluorescent staining of FANCD2 in GM847 cells treated with random or FANCD2 targeting siRNAs. FANCD2 foci are not observed in cells treated with siRNAs A-C.

FANCD2

FANCD2

A)

B) GM847 + random siRNA + FANCD2 siRNA D

+ FANCD2 siRNA A + FANCD2 siRNA B + FANCD2 siRNA C

71

GM847 and VA13 ALT cells, and GM639 and HT1080 telomerase positive cells were

transfected with FANCD2 siRNA A, then examined for telomere dysfunction induced foci

(TIFs) 5 days later. Transient FANCD2 depletion in GM847 and VA13 cells leads to a 2.9 fold

increase in the absolute number of 53BP1 foci that colocalize with TRF2, which corresponds to

2.9 and 3.1 fold increases in the percentage of TRF2 foci that have activated a DNA damage

response in GM847 and VA13 cells, respectively (Figure 2-10 A). GM639 and HT1080 cells do

not show any increase in 53BP1 colocalization with TRF2 when FANCD2 is depleted. Similar

increases in TIFs were also observed when GM847 and VA13 cells were treated with FANCD2

siRNA C (data not shown), and an ALT specific increase in TIFs upon FANCD2 depletion was

also reported by Spardy et al, (2008). In contrast to TIFs which form in an ALT specific manner,

FANCD2 depletion also results in an increase in 53BP1 foci not associated with TRF2, which is

observable in both ALT and telomerase positive cells. This increase in 53BP1 foci is consistent

with a role for FANCD2 in promoting genomic stability at non-telomeric regions.

Telomere shortening can trigger activation of a DNA damage response (d'Adda di Fagagna et al,

2003), and aberrant resolution of t-loops or breaks in the telomere sequence can trigger rapid

shortening events (Wang et al, 2004). Therefore I examined chromosomes during metaphase for

the presence of chromosomes ends without detectable telomeric signals. A constant exposure

time of 300 ms was used for all cell lines, and slides from each replicate were processed and

photographed on the same day. Five days after siRNA addition, FANCD2 knockdown did not

result in an increase in signal free ends in GM847 or U2OS ALT cells, or GM639 or HT1080

telomerase positive cells (Figure 2-10 B). In all cases >97% of chromatids had detectable

telomere signals. In GM847 and U2OS ALT cells this result was confirmed with a second

FANCD2 targeting siRNA, and again I found that >97% of chromosome ends in FANCD2-

depleted cells have telomere signals (Figure 2-10 B). Telomeres in FANCD2 depleted ALT cells

appear to have similar length distributions as controls (Figure 2-10 C) and sister chromatid

telomeres appear approximately equal in length, arguing against a severe replication problem,

although it remains possible that minor variations in telomere length distributions exist.

Together, my results suggests that telomeric DNA in ALT cells more frequently activates a DNA

damage response when FANCD2 is depleted, but that this is not due to telomere rapid deletion

events.

72

A)

B)

C)

Figure 2-10. FANCD2 knockdown results in an ALT-specific increase in telomere

dysfunction induced foci that is independent of telomere rapid deletion events

A) Plot of the number of 53BP1 foci that do, and do not colocalize with TRF2 from 300

randomly selected asynchronous cells from 3 independent experiments ± the associated standard

error. B) Plot of the average percentage of chromatid ends that do not have detectable telomere

GM639 GM639 HT1080 HT1080 VA13 VA13 GM847 GM847 +random +FANCD2 +random +FANCD2 +random +FANCD2 +random +FANCD2 siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA

si

GM639+ GM639+ HT1080+ HT1080+ GM847+ GM847+ GM847+ VA13+ VA13+ VA13+ random FANCD2 random FANCD2 random FANCD2 FANCD2 random FANCD2 FANCD2 siRNA siRNA A siRNA siRNA A siRNA siRNA A siRNA B siRNA siRNA A siRNA B

si

73

signals from 3 independent experiments ± the standard deviation of experimental means. C)

Representative metaphase spreads from U2OS cells treated with random or FANCD2 siRNA.

Telomeres (red) are present on almost all chromatids, and do not usually differ significantly in

size between sister chromatids. Centromeres are shown in green.

2.4.7 ALT-associated PML bodies (APBs) are structurally different from non-ALT bodies, and contain telomeric nucleic acid in the interior of the body that differs from surrounding chromatin

As the majority of FANCD2 colocalization with telomeric DNA and proteins occurs within

APBs, I wanted to better understand the nature of these structures. Non-ALT PML bodies have a

consistent and well defined ultrastructure: a solid protein core devoid of both DNA and RNA,

which makes frequent contacts with neighbouring chromatin fibers that help tether the body in

place (Boisvert et al, 2000). In contrast, fluorescent microscopy suggests that APBs have a

donut-shape that is closely associated with telomeric DNA within or around the body (Yeager et

al, 1999). Unfortunately, limitations in the resolution of light microscopes have precluded

precise determination of the ultrastructure and organization of APBs. A recent attempt to study

APB structure using light microscopy relied on experimentally inducing formation of abnormally

large APBs (Draskovic et al, 2009), however whether these larger APBs resemble the

endogenous bodies is unclear. APBs may serve as sites where ECTR DNA is sequestered, where

telomeres undergo replication or recombination events, and/or sites where late replicating

telomeric chromatin is remodeled.

Electron spectroscopic imaging (ESI) is a technique that monitors energy loss of incident

electrons as they pass through samples in order to generate high resolution element specific maps

(reviewed in Bazett-Jones et al, 2008). Through monitoring the respective ratios of nitrogen and

phosphorus one can determine whether structures are primarily composed of protein, DNA, or

RNA. Examination of the ultrastructure of APBs using ESI was performed by Andrew Larsen

and Dr. Ren Li in the laboratory of Dr. David Bazett-Jones. A combination of fluorescent

staining of TRF2 on embedded sections and gold labeling of PML, was used to distinguish APBs

from non-ALT associated PML bodies in GM847 and VA13 ALT cells. ESI analysis showed

that APBs have a unique ultrastructure, and clearly contain a mixture of protein (blue) and

nucleic acid (yellow) within the interior of the body, surrounded by an outer protein layer (Figure

74

2-11). Within the VA13 cell line, APBs were found that are similar to non-ALT PML bodies in

size (200-350nm), however examples of APBs that are significantly larger then non-ALT PML

bodies were also identified (Figure 2-11). All APBs examined exhibited a distinct ultrastructure

from non-ALT PML bodies, in that they clearly contained phosphorous, representing nucleic

acid, within the bodies. The intensity and morphology of the phosphorous signal differs

significantly from neighbouring chromatin, suggesting that APBs primarily contain non-

nucleosomal material. APBs in GM847 ALT cells have a similar structure as APBs in the VA13

ALT line, and were also found to contain phosphorous within the bodies that is less intense then

neighbouring chromatin (Figure 2-11). Gold labeling of PML suggested that the PML protein is

primarily concentrated within the outer protein layer surrounding the APB.

APBs in GM847 ALT cells treated with random and FANCD2 siRNA were also examined, and

found to have a similar ultrastructure as controls, with an outer protein shell surrounding a

nucleic acid/protein mixture. The intensity of the phosphorous signal within APBs in random

siRNA treated cells differs significantly from surrounding chromatin (Figure 2-12). APBs in

FANCD2-depleted GM847 cells resemble APBs from random siRNA treated cells with a few

notable exceptions. APBs in FANCD2-depleted GM847 cells tend to be physically larger then

APBs in random siRNA and untreated GM847 cells. Similar to controls, APBs in FANCD2-

depleted cells show a wide size distribution, however bodies tend to have diameters in excess of

half a micron, with some bodies being greater then one micron in size. APBs of this size were

not observed in untreated or random siRNA treated GM847 cells.

APBs were also observed in FANCD2-depleted GM847 cells that contain blocks of phosphorus

signal with an intensity and morphology similar to the surrounding chromatin (Figure 2-12).

Analysis of serial sections of several of these bodies indicated that these chromatin-like signals

were not simply on top or below the body, but rather had invaded the APBs. Given that

telomeres are highly nucleosomal structures, these chromatin-like masses may represent

telomeres themselves, or ECTR DNA derived directly from telomeres, associating with APBs

(Pisano et al, 2008). If these chromatin masses represent telomeres, this would suggest that

interactions between telomeres and APBs are usually rare or transient in nature, or do not occur

under normal circumstances, as they were not detected during the analysis of APBs in VA13 or

GM847 control cells.

75

VA13

VA13

VA13

GM847

Figure 2-11. APBs contain nucleic acid within the body, differing from non-ALT associated

PML bodies, which are solid protein structures Images of nitrogen, phosphorous, and a false coloured overlay image of nitrogen (blue) and phosphorous

(yellow) are shown for a non-ALT associated PML body (top row, solid arrow), and APBs (dotted arrow)

in VA13 (rows 2 &3) and GM847 (row 4) ALT cells. Scale bar is 0.2 µm. Unlike non-ALT associated

PML bodies that are devoid of phosphorous signal, APBs contain phosphorous within the body. The

intensity of the phosphorous within APBs differs significantly from neighbouring chromatin.

76

GM847 + random siRNA

GM847 + FANCD2 siRNA

GM847 + FANCD2 siRNA

Figure 2-12. APBs in FANCD2 depleted cells tend to be physically larger then APBs in

controls, and can contain blocks of chromatin-like nucleic acid Images of nitrogen, phosphorous, and a false coloured overlay image of nitrogen (blue) and phosphorous

(yellow) are shown for a large APB from a random siRNA treated GM847 cells (top row), and average

sized APBs in FANCD2-depleted GM847 cells (bottom 3 rows). Scale bar is 0.2 µm. Examples of APBs

with chromatin-like structures within FANCD2-depleted APBs are shown (bottom 2 rows). Solid arrows

indicate APBs, dotted arrows indicate chromatin-like structures invading APBs.

GM847 + FANCD2 siRNA

77

2.4.8 FANCD2 depletion in ALT cells results in nuclear abnormalities, centrosome amplification and rapid cell death

Knockdown of FANCD2 in ALT cells results in the accumulation of cells with severe nuclear

abnormalities in GM847, VA13, SAOS-2, and GM637 ALT cells (Figure 2-13A, B; SAOS-2

and GM637 data not shown). These types of highly fragmented nuclei are not common in

telomerase positive cell lines depleted of FANCD2 and have not been reported in cells from FA

patients. I scored 600 interphase nuclei over 3 independent experiments for the presence of

micronuclei, holes, bridging, irregular multiple lobes and multiple nuclei and found that in cells

treated with FANCD2 siRNA A, 6.3 ± 1.4% of GM639 and 3.0 ± 0.5% of HT1080 telomerase

positive cells were abnormal 5 days post-transfection, while 56.3 ± 6.5% of GM847 and 40.5 ±

7.9% of VA13 FANCD2-depleted ALT cells were abnormal (Figure 2-14B). Fragmented nuclei

of FANCD2-depleted ALT cells are not apoptotic by TUNEL assay, and can incorporate BrdU,

suggesting these cells are not terminally arrested in G1/G0 (data not shown).

SiRNA depletion of TRF2 causes similar types of nuclear abnormalities in GM847 and VA13

ALT cells (Figure 2-13 B), and VA13 ALT cells with uncapped telomeres have previously been

reported to form similar nuclei (Guiducci et al, 2001). This suggests that, that within these cell

lines, telomere dysfunction is one potential cause of this these types of abnormalities. U2OS

ALT cells that are depleted of FANCD2 do not show severe nuclear abnormalities, however this

cell line has wild type p53 and therefore may arrest in G1/G0 in response to telomere

dysfunction. Analogous types of abnormalities are observed in cells with aberrations in

kinetochore components, or mitotic spindle abnormalities in conjunction with a nonfunctional

spindle assembly checkpoint, suggesting that this may be a common phenotype of cells with

mitotic segregation problems (Goshima et al, 2003; Hauf et al, 2003; Taylor and McKeon 1997).

Additionally, I find that FANCD2 depleted GM847 ALT cells show increased frequency of

polyploidy and endoreduplication, abnormalities that are not increased in HT1080 telomerase

positive cells depleted of FANCD2. When endoreduplication is observed in GM847 FANCD2

depleted cells, it affects all chromosomes, suggesting that the entire genome has duplicated

without proceeding through mitosis. Depletion of FANCD2 results in an approximately 5 fold

increase in the frequency of GM847 ALT cells with a greater then 8N chromosome content as

determined by FISH analysis of centromosome numbers in interphase cells. In random siRNA

treated cells analyzed 5 and 8 days post siRNA addition, nuclei with a greater then 8N DNA

78

Figure 2-13. FANCD2 depletion results in severe nuclear abnormalities in GM847 and

VA13 ALT cells A) Examples of nuclear abnormalities frequently observed in FANCD2-depleted ALT cells. Scale bar is

10µm. B) Representative images of nuclei in GM847 and VA13 cells treated with random, FANCD2 or

TRF2 siRNA. Depletion of either FANCD2 or TRF2 results in abnormal nuclei. Random and FANCD2

depleted cells were imaged 5 days post siRNA addition, TRF2 depleted cells were imaged 72 hours post

siRNA addition due extensive cell death past this time point.

A)

B)

79

content occurred at frequencies of 3.5 ± 1.1% and 3.3 ± 1.1%, respectively. This differs from

FANCD2 siRNA treated cells analyzed 5 and 8 days post siRNA addition, that accumulate

nuclei with a greater then 8N DNA content at frequencies of 18.3 ± 2.4% and 19.3 ± 2.5%,

respectively. In HT1080 cells treated with random or FANCD2 siRNA, the frequency of nuclei

with a greater then 8N DNA content was <1% at all time points. Together these data suggest that

ALT cells depleted of FANCD2 face challenges during mitosis that may not occur in FANCD2-

depleted telomerase positive cells, difficulties that may be related to telomere dysfunction.

When more than two centrosomes are present in a cell, establishment of bipolar spindles may be

hindered leading to impaired chromosome segregation and aneuploidy. Cells with more then 2

centrosomes (γ-tubulin) foci are increased 5.1 and 28.1 fold over controls in FANCD2 depleted

VA13 and GM847 cells, respectively (Figure 2-14 A, B). Depletion of FANCD2 in GM639 and

HT1080 telomerase positive cells led to respective 1.6 fold increase and 0.76 fold decrease in the

frequency of FANCD2 depleted cells with supernumerary centrosomes relative to controls

(Figure 2-14 B). Potential causes of supernumerary centrosomes include targeted amplification

following DNA damage, cytokinesis failure or centrosome fragmentation. Given that FANCD2

depletion results in multinuclear cells and increased polyploidy in GM847 ALT cells, failed

cytokinesis may contribute to the observed supernumerary centrosomes. However, as only ∼19%

of FANCD2-depleted GM847 have a greater then 8N DNA content, but ∼47% cells have more

then 2 centrosomes, cytokinesis failure cannot be the sole cause of the increase. Centrosome

amplification has previously been observed in cells with uncapped telomeres (Guiducci et al,

2001) and other forms of genomic DNA damage (Bourke et al, 2007; Saladino et al, 2009),

suggesting that increased levels of DNA damage in FANCD2-depleted ALT cells may contribute

to the observed centrosome amplification.

The DNA repair factor BRCA1 has been localized to centrosomes (Parvin JD, 2009), where it is

thought to play a direct role in the regulation of centrosome stability and duplication. Using

indirect immunofluorescene I investigated whether FANCD2 also localizes to centrosomes.

FANCD2 localization to centrosomes was observed with a rabbit polyclonal antibody to

FANCD2, but not with a murine monoclonal antibody (data not shown). However, the observed

localization of FANCD2 to centrosomes persisted in FANCD2-depleted cells and FANCD2

patient cell lines, suggesting that staining is an artifact of the antibody (data not shown). These

80

abnormal nuclei

>2 centrosomes

results, in conjunction with the largely ALT specific nature of the centrosome amplification,

argue against a direct role for FANCD2 in centrosome stability. Taken together, my results

suggests that the observed centrosome amplification in FANCD2-depleted ALT cells is due to a

combination of the mitotic and cytokenesis failures, as well as targeted amplification of

centrosomes in cells with high levels of DNA damage.

A) GM847 + FANCD2 siRNA

B)

Figure 2-14. FANCD2 depletion increases the frequency of abnormal nuclei and cells with

supernumerary centrosomes in GM847 and VA13 ALT cells, but not in GM639 and

HT1080 telomerase positive cells A) Representative images of GM847 cells treated with FANCD2 siRNA with more then 2 centrosomes.

γ-tubulin (red) was used as a centrosome marker. Scale bar is 10 µm. B) Plot of the number of abnormal

nuclei (purple) and nuclei with more than 2 centrosomes (orange) from populations of 600 randomly

selected asynchronous cells over 3 independent experiments ± the associated standard error.

γ−tubulin

GM639+ GM639+ HT1080+ HT1080+ GM847+ GM847+ VA13+ VA13+ random FANCD2 random FANCD2 random FANCD2 random FANCD2 siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA

si

81

When ALT cells are depleted of FANCD2 there is a significant decrease in both short and long-

term viability. GM847 cells and U2OS cells were treated with FANCD2 siRNAs A-D and then

the remaining cell number was determined 5 days post siRNA addition. Similar to GM847 cells,

I find that FANCD2 protein levels in U2OS cells are significantly decreased by FANCD2 siRNA

A-C, but not D (Figure 3-1). Phase contrast images of the cell density 5 days post siRNA are

shown in Figure 2-15. The number of FANCD2 siRNA treated GM847 cells relative to random

siRNA treated cells 5 days post siRNA addition from 3 independent experiments was 19.6 ±

7.7%, 18.3 ± 3.8%, 33.1 ±4.8%, and 111.2 ± 26.2%, for cells treated with FANCD2 siRNA A-D,

respectively. In U2OS cells the number of FANCD2 siRNA treated cells relative to random

siRNA treated cells was 42.2 ± 6.7%, 31.2 ± 6.1%, 49.9 ± 14.5%, and 148 ± 43.5% for cells

treated with FANCD2 siRNAs A-D, respectively.

Figure 2-15. FANCD2 depletion leads to a decrease in cell growth and survival in ALT cells Representative phase contrast images of 847 and U2OS cells treated with random or FANCD2 targeting

siRNAs. Images were taken 5 days post siRNA addition at 5x magnification.

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To determine the long-term fate of FANCD2 siRNA treated cells, cells treated with FANCD2

siRNA A were replated for colony forming assays 4 days post siRNA transfection. FANCD2

begins to be re-expressed 8-9 days post siRNA addition, and was not added again after the initial

treatment, therefore the ability of cells to recover from damage incurred during their growth over

a one week period with reduced FANCD2 levels was assessed. Colony forming ability was

reduced relative to random siRNA treated cells in GM847, U2OS, and GM637 ALT cells. VA13

and SOAS-2 ALT cells were not included in this analysis because FANCD2-depleted cells failed

to form colonies at any of the cell numbers plated. The colony forming ability of GM847,

GM637, and U2OS FANCD2 depleted cells was determined relative to random siRNA treated

cells over 4 independent experiments, and was 1.5 ± 1.2%, 16.0 ± 17.1%, and 20.8 ± 30.2%,

respectively. Together these results suggest that FANCD2 plays an essential role in promoting

ALT telomere maintenance and short and long term cell viability.

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2.5 Discussion

In this study I found that FANCD2 and FA core complex proteins FANCA and FANCG

colocalize with telomeric foci in human cells that use the ALT pathway, but not in primary cells

or cells that rely on telomerase for telomere maintenance. In addition to colocalization, FANCD2

coimmunoprecipitates with TRF2 and BLM in ALT cells and FANCD2 depletion leads to an

ALT-specific increase in TIFs. Similar observations of largely ALT-specific roles in telomere

maintenance have also been made for MUS81, topoisomerase IIIα and BLM (Zeng and Yang,

2009; Temime-Smaali et al, 2008; Bhattacharyya et al, 2009). One hypothesis for the unique

requirement of certain DNA repair factors in ALT, is that they are involved in the response to

dysfunctional telomeric DNA, which is present at a higher level in ALT then telomerase positive

cells. However, my data shows that most FANCD2 localizes to telomeric foci that have not

activated a DNA damage response. Additionally, I have examined late passage primary cells

with a high frequency of short and dysfunctional telomeres do not see frequent FANCD2

colocalization with telomeric foci (data not shown). This suggests that there is something unique

to the ALT situation that the FA pathway is responding to.

FANCA and FANCG primarily colocalize with FANCD2 within larger telomeric foci, which

most likely represent APBs. FA core complex proteins are typically difficult to visualize by IF,

presumably because they associate with DNA damage at very low abundance or transiently. A

recent study on telomeric DNA in ALT cells shows the existence of the following species of

telomeric DNA: gapped double-strand, single-strand, circular, and complex branched structures

with a mixture of single- and double-strand DNA (Nabetani and Ishikawa, 2009). Fractionation

of nuclear lysate shows substantial fractions of these species exist as extra-chromosomal DNAs,

which as discussed below, appear to accumulate within APBs (Nabetani and Ishikawa, 2009).

The FA pathway can be activated by a variety of DNA substrates with branched structures and

exposed ends (Sobeck et al, 2007) and purified human FANCD2 has highest in vitro binding

affinity to ssDNA, followed by splayed arms, Holliday junction structures, and dsDNA (Roques

et al, 2009). Taken together, these observations suggest that the ability to clearly visualize FA

core complex proteins in ALT cells may be due to unusually high local concentrations of

substrates for FA core complex binding found within APBs. Stoichiometric differences in the

84

local concentration of DNA structures that recruit FANCD2 may also explain why FANCA

knockdown efficiently reduces non-telomeric FANCD2 foci, but FANCD2 foci colocalized with

telomeric DNA remain weakly visible. During replication FANCD2 may accumulate at single

spatially distinct lesions, whereas APBs may contain high numbers of DNA molecules that bind

FANCD2 within a single focus.

Large APBs found in interphase ALT cells are often postulated to primarily contain multiple

ALT telomeres undergoing replication and recombination reactions, however direct evidence of

this is lacking. Recently, Draskovic and colleagues expressed a modified form of Herpes simplex

virus protein ICP0 to generate abnormally large APBs, and found that subtelomeric sequences

frequently associate with the modified APBs (Draskovic et al, 2009). Whether these

subtelomeric sequences can accumulate in the ECTR at levels detectable by FISH was not tested,

raising questions over the suitability of these sequences as a marker for telomeres. Additionally,

immunofluorescent analysis of the enlarged APBs suggested that the bodies are solid protein

structures, with telomeric DNA clustered exclusively around the periphery of the body

(Draskovic et al, 2009). APBs with a solid protein core were never observed in our study of

endogenous APBs, and I hypothesize that expression of the modified ICP0 protein significantly

disrupted normal APB structure.

Another recent study by Jegou and colleagues, showed that lacO sequences integrated near the

telomeres of chromosomes associates with PML bodies, however results must be cautiously

interpreted, as the distance between the integration site and telomere was not determined, and it

is not clear whether this observed colocalization with PML is affected by local changes in

chromatin structure caused by the integration of 0.7-3.7 Mb arrays of lac O repeats (Jegou et al,

2009). Immunofluorescent analysis of lac O repeats argues for a model where PML frequently

forms a large cap-like structure that engulfs telomeric chromosomal DNA (Jegou et al, 2009).

Electron spectroscopic imaging of endogenous interphase APBs argues against this idea, because

although APBs contain nucleic acid in the interior of the body, the structure of the material and

phosphorous signal intensity are inconsistent with chromatin. Our data suggests that APBs

primarily contain non-nucleosomal ECTR DNA.

85

It remains possible that nucleic acid within these APBs represents ALT telomeres which have an

abnormal organization that does not resemble normal chromatin, however γH2AX has been

detected at ALT telomeres during mitosis, arguing that, similar to telomeres in telomerase

positive and primary cells, ALT telomeres are nucleosomal structures (Cesare et al, 2009).

Additionally, in FANCD2-depleted ALT cells, APBs with chromatin-like masses invading the

bodies were observed. If these masses represent telomeres, then it would suggest that interactions

are either FANCD2-depletion specific or are normally infrequent or transient in nature in wild-

type interphase cells, and therefore the study of APBs ALT cells is largely the examination of the

ECTR DNA and its associated proteins.

The idea that APBs are primarily composed of ECTR is supported by studies showing that

treatment of ALT cells with DNA-damaging agents results in concomitant increases in ECTR

DNA and APBs (Fasching et al, 2007), and during metaphase most APBs present within cells are

clearly extra-chromosomal (Nabetani et al, 2004). Additionally non-ALT cells with elongated

telomeres can accumulate ECTR DNA and APBs, but do not show an increase in TIFs or

recombination events involving telomeres (Pickett et al., 2009), and a subclone of the VA13

ALT line was identified which no longer has ECTR DNA or APBs, but continues to have an

elevated frequency of recombination among telomeres (Cerone et al., 2005). The above studies

suggest that while there is a close connection between the generation of ECTR DNA and the

formation of APBs, APBs are not essential for most recombination reactions involving

telomeres. Strand specific FISH suggests that many recombination events between telomeres in

ALT cells may be reciprocal exchanges between sister chromatids, a form of recombination

which can likely occur outside of large ALT-specific subnuclear domains (Bailey et al, 2004;

Londoño-Vallejo et al, 2004).

While APBs may not be required for telomeric recombination in ALT cells, APBs do appear to

play an important role in ALT, as they are present to varying degrees in almost all ALT cells,

and they arise at the same time as activation of the ALT pathway (Yeager et al, 1999). Data

presented in this study supports the hypothesis proposed by Fasching and colleagues (2007), that

APBs are primarily involved in the sequestering of ECTR DNA. ECTR DNA generated in ALT

cells has been shown to have frequent internal ssDNA regions and exposed ends (Nabetani and

Ishikawa, 2009), substrates that have the potential to activate DNA damage response pathways

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and cell cycle checkpoints. Although ALT appears to be exclusively active in cancer cells and

immortalized cell lines which frequently have mutations in the p53 pathway, experiments in

ALT cells exposed to exogenous forms of DNA damage have shown that DNA damage

responses and cell cycle checkpoints remain functional (Wang et al, 2002; Demuth et al, 2008;

Cliby et al, 1998). This raises the question of how ALT cells manage to cycle in the presence of

endogenous DNA substrates, such as ECTR DNA, that would be predicted to elicit a DNA

damage and checkpoint response. One possibility would be that ECTR DNA is sequestered in

APBs away from components of the DNA damage sensors to avoid activation or the DNA

damage response. However this model does not fit with the observation that APBs are present in

viable cells that are cycling, yet APBs can associate with DNA damage sensors and DNA repair

factors that have been phosphorylated by ATM and/or ATR (Grobelny et al, 2000; Nabetani et

al, 2004; Wu et al, 2003; Stagno D'Alcontres et al, 2007). The presence of proteins

phosphorylated by ATM and ATR also argues that components of the shelterin telomere binding

complex localized to APBs cannot fully suppress activation of the DNA damage response

pathway by ECTR DNA (Stagno D'Alcontres et al, 2007), even though they substantially

attenuate this response at telomeres and other genomic loci following ionizing radiation in

telomerase positive and primary cells (Denchi and de Lange, 2007; Karlseder et al, 2004;

Bradshaw et al, 2005).

I instead hypothesize that APBs may serve as essential sites where DNA that has activated a

damage response, and associated DNA repair factors, are targeted. The local environment within

and around APBs may be non-conducive to propagation of the DNA damage/checkpoint

response, or alternately may serve as a region where the DNA damage response is actively

down-regulated. Sequestering ECTR DNA within APBs may prevent cells from fully activating

cell cycle checkpoints, or lead to the eventual down-regulation of any activated checkpoints. A

recent analysis of γH2AX staining patterns in ALT cells arrested in metaphase with colcemid

demonstrates that cells can enter mitosis with telomeres that have activated a DNA damage

response, and that some of these telomeres are located in close proximity to APB-like structures

(Cesare et al, 2009). It is possible that dysfunctional telomeres must associate with APBs to

satisfy the G2/M checkpoint, allowing cells to proceed into mitosis, than move away from the

bodies during segregation. Alternately, dysfunctional telomeres closely associated with APB-like

87

structures may represent a later form of an interphase phenomenon that is occasionally observed

using electron microscopy in FANCD2-depleted cells.

Transient depletion of FANCD2 results in the concomitant increase in telomeric DNA that has

activated a damage response, and the detection of APBs with chromatin masses tightly

associating with the bodies. The chromatin structures may represent dysfunctional telomeres,

which may associate with APBs, and interact with ECTR DNA or additional telomeres, if

present at the same time at the bodies. If difficulties arise in resolving interactions prior to

entering mitosis, telomeres may remain closely associated with APBs during mitosis, similar to

what was observed by Cesare and colleagues. Whether dysfunctional telomeres can be directly

targeted to APBs, or interact more frequently as an indirect result of the increased mobility of

dysfunctional telomeres remains to be resolved (Dimitrova et al, 2008).

The molecular details of the role of FANCD2 within ALT remains under investigation, however

my study of the requirements for FANCD2 localization to telomeric foci and APBs suggest that

FANCD2 is not just participating in the response to stalled/collapsed replication forks or

interstrand crosslinks, as the localization of FANCD2 is largely ATR independent. Additionally,

localization is independent of ATM, and most FANCD2 localizes to telomeric foci that do not

contain 53BP1, suggesting that FANCD2 is not simply localizing to telomeric DNA ends that

have activated a DNA damage response. Interestingly, I observe a unique requirement for BLM

expression in order for FANCD2 to localize to ALT telomeric foci. Although there is a growing

body of evidence suggesting that FANCD2 and BLM participate in common pathways, (Chan et

al, 2009; Naim and Rosselli, 2009; Pichierri et al, 2004; Hemphill et al, 2009) ALT appears to be

the first situation where BLM is acting upstream of FANCD2.

BLM may be directly involved in promoting FANCD2 localization to telomeric DNA, or

alternately may be required to generate a DNA substrate that FANCD2 subsequently then binds

to. Telomeres present several unique challenges during DNA repair and replication because of

the potential tendency of the TTAGGG repeat to form secondary structures and G-quadruplexes,

structures that BLM may be required to resolve (Mohaghegh et al, 2001). Supporting the idea

that BLM plays key roles in ALT, knockdown of BLM leads to a rapid, ALT-specific decrease in

average telomere length (Bhattacharyya et al, 2009), and when I deplete BLM, I no longer

observe large APBs with high amounts of TRF1, TRF2, and telomeric DNA, suggesting that

88

there may be a decrease in production of ECTR DNA (data not shown). Additional experiments

presented in the following chapter suggest that BLM acts both up and downstream of FANCD2,

and that a key role of FANCD2 within ALT is in the regulation of BLM-dependent telomeric

recombination and ECTR DNA synthesis.

FANCD2 also appears to be involved in preventing ALT telomeric DNA from activating a DNA

damage response, as FANCD2-depletion leads to an ALT specific increase in TIFs. One cause of

TIFs could be an increase in collapsed replication forks, however this seems unlikely because

most FANCD2 does not appear to be responding to stalled/collapsed forks, as FANCD2

recruitment to telomeric foci is largely ATR independent. However, FANCD2 may have

additional, currently unknown functions during replication, which are required to prevent

activation of a DNA damage response. Supporting this idea, spontaneous FANCD2 foci that

normally form during S phase are also independent of both ATM and ATR.

Critically short telomeres can activate a DNA damage response (Zou et al, 2004), however my

analysis of telomere free ends demonstrates that FANCD2 depletion does not alter the frequency

of chromosome ends without a detectable telomere signal. It should be noted that the level of

telomere free ends I find differs markedly from recent findings of Fan et al., (2009) who reported

that 45% of telomeres were undetectable in FANCD2 depleted U2OS cells. The reason for this

difference is unknown, however may be related to technical differences in FISH sensitivities and

the decision of Fan et al. to vary camera exposure times between experimental conditions.

Supporting the idea that my FISH experiments were more sensitive then those by Fan and

colleagues, the baseline level of telomere free ends reported by Fan and colleges was very high

(∼18% in U2OS cells and ∼35% in 847 cells), whereas I find that only ∼2% of telomeres are

undetectable under all conditions, a figure more consistent with previously reported values of

∼5% of GM847 chromosomes having a telomere free end (Zeng et al, 2009; Perrem et al, 2001).

Additionally, although Fan et al, observed a dramatic level of signal free ends in FANCD2

depleted cells via microscopic detection of telomeric signals, the proportion of the population

with lower amounts of total telomeric DNA did not increase in flow cytometric measurements,

indicating a technical problem with microscopic FISH.

89

Evidence suggests that factors in addition to telomere length may influence the ability of

telomeres to suppress the DNA damage response. ALT metaphase chromosomes with strong

telomeric signals can activate a DNA damage response (Cesare et al, 2009). Proper capping of

telomeres is also likely influenced by formation of secondary DNA structures, as well as

sufficient quantities of components of the shelterin telomere binding complex. Both t-loops and

components of shelterin are present in ALT and telomerase positive cells (Cesare and Griffith,

2004) making it difficult to understand why FANCD2-depletion would result in an ALT-specific

capping problem. Additionally, proteins outside of shelterin that have been implication in

capping, such as the recombinational protein RAD51D, localizes to telomeres in telomerase

positive and ALT cells, and cause telomere abnormalities in both cell types when depleted

(Tarsounas et al, 2004).

One hypothesis that may explain the ALT-specific FANCD2 dependent increase in TIFs relates

to the presence of single-strand gapped regions within ALT telomeric. ALT telomeric DNA

appears to differ from non-ALT telomeric DNA, both at telomeres and extra-chromosomally, in

that there is an elevated frequency of internal ssDNA gapped regions (Nabetani and Ishikawa,

2009). FANCD2 has recently been demonstrated to bind preferentially to ssDNA in vitro,

although its function at ssDNA remains unclear (Roques et al, 2009). The ability of FANCD2 to

suppress TIFs may relate to the ability of FANCD2 to bind to gapped regions and regulate the

subsequent recruitment of down stream factors such as ATR/ATRIP, BRCA2, and RAD51.

When FANCD2 is depleted, ssDNA gaps present in ALT telomeric DNA may be more likely to

activate a DNA damage response, leading to an increase in TIFs. An alternate hypothesis related

to insufficient amounts of telomere binding proteins due to amplification of ECTR DNA in

FANCD2-depleted cells will be discussed in the following chapter.

The increase in TIFs in FANCD2-depleted ALT cells may be a contributing factor to the nuclear

abnormalities and centrosome amplification observed. Introduction of double-strand breaks

through ionizing radiation, prolonged replication stress, or topoisomerase II inhibitors leads to

targeted centrosome amplification during an extended G2 phase in human cell lines (Bourke et

al, 2007; Saladino et al, 2009). This suggests that centrosome amplification represents an

apoptosis-independent mechanism of targeting damaged cells for death, because most cells will

undergo an abnormal mitotic division and die during the subsequent cell cycle. Additionally,

90

when telomeres are uncapped in VA13 ALT cells, supernumerary centrosomes are observed

(Guiducci et al, 2001), demonstrating that TIFs are another form of damage that cause

centrosome amplification.

Data presented in this study suggests that the role of the FA pathway in telomere maintenance

may be largely limited to cells that utilize the ALT pathway. As ALT has only been shown to be

active in cancer cells and immortalized cell lines, it is unlikely that telomere problems are a

driving force in the pathogenesis of FA. However, because the FA pathway appears to be

essential for ALT cell viability, treatment of ALT positive cancers with small molecule inhibitors

of the FA pathway may prove to be a viable therapeutic approach. Although the overall fraction

of tumors that utilize the ALT pathway appears to be only ∼10%, ALT is activated more

frequently in tumors of neuroepithelial and mesenchymal origin suggesting that for some cancers

targeting of the FA pathway may be a viable therapeutic approach (Henson et al, 2005; Costa et

al, 2006). ALT also may serve as a useful tool for deciphering the molecular function of the FA

pathway in recombination and repair, as ALT cells have an unusually high number of

endogenous substrates that activate and recruit pathway components.

91

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Chapter 3

3 Fanconi Anaemia Protein D2 Limits BLM-Dependent, RAD51-Independent Telomeric Recombination and DNA Synthesis in ALT-Immortalized Human Cells

3.1 Abstract

The alternative lengthening of telomeres (ALT) pathway is a recombinational telomere

maintenance pathway active in a fraction of immortalized cell lines and cancer cells. BLM, the

Bloom syndrome helicase, plays an essential role in ALT, as overexpression of BLM results in

rapid synthesis of telomeric DNA, and depletion of BLM results in telomere shortening

(Stavropoulos et al., 2002; Bhattacharyya et al., 2009). BLM associates with multiple

components of the Fanconi Anaemia (FA) pathway, (Meetei et al, 2003; Pichierri et al, 2004;

Deans and West, 2009), a rare inherited syndrome characterized by genomic instability, bone

marrow failure, and cancer predisposition. I have previously shown that both FA core complex

components and FANCD2 localize to telomeric foci and ALT associated PML bodies (APBs),

and that localization of FANCD2 to telomeric foci depends on both the monoubiquitination of

FANCD2 by the FA core complex, and the expression of BLM. Here, I now report data

suggesting that FANCD2 plays a critical role in human ALT cells by limiting BLM-dependent

telomeric recombination and amplification events. Transient depletion of FANCD2 results in a

rapid ALT-specific increase in telomeric DNA synthesis, analogous to the phenotype caused by

overexpression of BLM (Stavropoulos et al., 2002). Excess telomeric DNA synthesized in

FANCD2-depleted ALT cells appears to be primarily extra-chromosomal, and accumulates both

outside of and within APBs. Electron spectroscopic imaging of APBs in FANCD2-depleted cells

shows that most bodies are physically larger and contain more nucleic acid then control APBs,

and RNase treatment demonstrates that the material within bodies is DNA, not RNA. FANCD2

depletion also results in an increase in telomeric DNA entanglements, expression of fragile site

characteristics at telomeres, telomere sister chromatid exchanges, and localization of RAD51 to

telomeric foci in ALT cells. Telomere abnormalities in FANCD2-depleted cells are completely

rescued by codepletion of BLM, but not RAD51. I propose that while BLM is required to

promote recombination and replication of telomeric DNA in ALT cells, this activity is regulated,

and that FANCD2 plays a critical role in this regulation.

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3.2 Introduction

Maintenance of genome stability requires the concerted effort of numerous factors to ensure

accurate repair, replication and segregation of chromosomes. Members of the RecQ family of

DNA helicases play important roles in all these processes, and are known as ‘caretakers of the

genome’ because of their role in preventing genomic instability (reviewed in Chu and Hickson,

2009; Bachrati and Hickson, 2008). While unicellular organisms typically express a single RecQ

helicase, human cells express at least five different RecQ helicases (RECQ1, BLM, WRN,

RECQ4, RECQ5), of which BLM, WRN, and RECQ4 have been implicated in human

syndromes associated with elevated cancer incidence (Ellis et al, 1995; Yu et al, 1996, Kitao et

al, 1999, Siitonen et al, 2003; Siitonen et al, 2009). BLM is mutated in Bloom syndrome, a rare

recessive disorder characterized by predisposition to a wide range of cancers, sun sensitivity, and

short stature (German J, 1993). Bloom syndrome cells display elevated levels of sister chromatid

exchanges and other chromosomal abnormalities including increased chromatid breaks, gaps,

radials, telomere associations, anaphase bridge and lagging chromosomes (Chaganti et al, 1974;

German and Crippa, 1996, Lillard-Wetherell et al, 2004). Similar to other RecQ mutants, BLM

cells are also sensitive to exogenous damaging agents, suggesting that RecQ helicases play

essential roles in the response to spontaneous and induced DNA lesions (Hemphill et al, 2009;

Aurias et al, 1985).

In addition to roles in promoting global genomic stability, RecQ helicases also can play essential

roles in maintaining telomeres. In yeast, mutations in genes encoding telomerase components

lead to cell death, with rare survivors emerging that rely on telomerase-independent pathways to

maintain telomeres. Type I survivors display amplification of the subtelomeric Y’ element, with

only a short terminal telomere repeat track (Lundblad and Blackburn, 1993), while type II

survivors have long tracts of telomere repeats (Teng and Zakian ,1999). Both types of survivors

rely on recombination, as they are dependent on Rad52p expression, however additional genetic

requirements suggest that the pathways are mechanistically distinct. Type II recombination

appears to function via a Rad51-independent mechanism, which requires expression of the RecQ

helicase Sgs1p, and Rad50p, a component of the MRX complex (Le et al, 1999; Huang et al,

2001).

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Telomeres in yeast type II survivors share many characteristics with telomeres in human cells

that have activated the Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway

also appears to function via recombination (Dunham et al, 2000; Londoño-Vallejo et al, 2004;

Muntoni et al, 2009), and similar to most type II survivors, ALT cells are characterized by the

presence of unstable telomeres with long tracts of telomeric repeats (Bryan et al, 1995). ALT

cells also contain circular and linear Extra-Chromosomal Telomeric Repeat DNA (ECTR),

which may be a product as well as a substrate in telomeric recombination reactions. Circular

ECTR DNA has been suggested to be involved in a ‘roll-and-spread’ mechanism of telomere

elongation in type II yeast survivors, where it can act as a template in rolling circle replication,

allowing for rapid increases of telomere length (Natarajan and McEachern, 2002; Lin et al,

2005). The known genetic requirements of ALT are also similar to type II recombination, with

both NBS1, a component of the MRE11/RAD50/NBS1 complex, and BLM being important for

maintaining telomere length (Zhong et al, 2007; Bhattacharyya et al., 2009).

Telomeric DNA in ALT cells has been reported to have frequent internal single-strand breaks or

gaps, which may arise due to incomplete replication or processing of stalled replication forks,

suggesting that ALT telomeric DNA is difficult to replicate (Nabetani and Ishikawa, 2009).

Telomeres in telomerase positive cells do not normally show ssDNA regions (Nabetani and

Ishikawa, 2009), however when confronted with replication stress or loss of TRF1, a key

telomere binding protein, telomeres in these cells exhibit an interrupted staining pattern via FISH

that may be due to ssDNA regions, and that is also observed in other difficult to replicate

sequences known as fragile sites (Sfeir et al, 2009). Interestingly, the frequency of telomeres

with abnormal staining patterns increases in BLM deficient cells, suggesting that BLM plays a

role in promoting replication of telomeric DNA (Sfeir et al, 2009). The involvement of BLM at

telomeres may be partially related to its ability to resolve non B-Form DNA structures (Sun et al,

1998), an activity which may be important because of the capacity of the telomeric G-strand to

promote G quadruplex structures that must be resolved prior to replication or recombination.

Although RecQ helicases clearly play beneficial roles at the telomere, evidence also suggests that

RecQ helicases can promote events that are ultimately deleterious to the cell. Fission yeast

lacking the telomere binding protein Taz1, have a telomere hyper-recombination phenotype,

accompanied by telomere entanglements, chromosome missegregation and loss of viability when

grown at lower temperatures (Miller and Cooper, 2003). These telomere abnormalities appear to

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arise during replication via a process promoted by Rqh1, the Schizoscaccharomyces pombe

RecQ helicase (Miller and Cooper, 2003; Rog et al., 2009). Furthermore, in Taz1Δ cells also

lacking telomerase, Rqh1 mediates a rapid telomere loss phenotype. However Rqh1 is ultimately

required to promote survivors that use an ALT-like telomere maintenance mechanism (Rog et

al., 2009). In the human ALT system, BLM also appears to have a dual role where it is required

to prevent telomere shortening (Bhattacharyya et al., 2009), and yet if BLM is overexpressed

there is a rapid increase in telomeric DNA synthesis followed by cell death (Stavropoulos et al.,

2002). Together these results suggest that RecQ helicases play a pivotal role in promoting

replication and recombination reactions at telomeres, but these reactions can be potentially

detrimental and therefore must be regulated. In fission yeast, sumoylation of Rqh1 is involved in

regulation of its activity at dysfunctional telomeres (Rog et al., 2009), and in this study I provide

evidence that FANCD2, a protein defective in Fanconi anaemia, regulates BLM dependent

telomeric replication and recombination reactions in human ALT telomere maintenance.

Fanconi anaemia (FA) is rare multigenic syndrome characterized by bone marrow failure, cancer

predisposition and congenital abnormalities. Protein products of FA genes can be divided in

three major categories; proteins which are essential components of a core complex with E3

ubiquitin ligase activity (FANCA, B, C, E, F, G, L), proteins that are monoubiquitinated by the

FA core complex (FANCD2, I), and proteins which have roles outside of ubiquitination

(FANCD1/BRCA2, FANCJ/BACH1, FANCM). Monoubiquitination of FANCD2 and FANCI

occurs during replication and following exposure to DNA damaging agents, and is required for

protein accumulation in nuclear foci and on chromatin (Garcia-Higuera et al, 2001;

Smogorzewska et al, 2007). Similar to Bloom syndrome, FA cells show signs of genomic

instability, with elevated frequencies of spontaneous chromatid breaks, gaps, and radials,

however the pattern of spontaneous rearrangements appears different in cells from the two

syndromes, suggesting that the underlying cause may differ (Schroeder and Kurth, 1971;

Schroeder and German, 1974). FA cells are also hypersensitive to interstrand crosslinking agents

and form radials at an elevated frequencies following treatment, which is the confirmatory

diagnositic criteria of FA (Auerbach et al, 1979). Interestingly, BLM deficient cells are also

sensitive to interstrand crosslinks, show increased radial formation following treatment, and

genetic evidence shows that BLM functions in the same pathway as FA proteins in response to

crosslinks (Pichierri et al., 2004; Hirano, 2005; Hemphill et al, 2009).

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I, and others, have found that FANCD2 associates with telomeric foci specifically in human cells

that utilize the ALT pathway, and that this localization depends on monoubiquitination by the

FA core complex (Spardy et al, 2008; Fan et al, 2009). Interestingly, I also find that FANCD2

coimmunoprecipitates with BLM in late S/G2 ALT cells, FANCD2 almost always localizes to

telomeric foci that also contain BLM, and BLM expression is required for FANCD2 localization

to telomeric foci. An interaction between FANCD2 and BLM has been reported in a study on

DNA repair (Pichierri et al., 2004), and FANCD2 was recently reported to localize to the base of

ultra-fine DNA bridges coated with BLM during mitosis, however the functional relationship

between these two proteins has remained unclear (Chan et al, 2009; Naim et al, 2009).

In this study I have continued to investigate the role of FANCD2 in ALT telomere maintenance

and find that transient depletion of FANCD2 results in a rapid ALT-specific increase in

telomeric DNA synthesis, analogous to the effect observed when BLM is transiently

overexpressed (Stavropoulos et al., 2002). The majority of telomeric DNA synthesized in

FANCD2-depleted ALT cells appears to be extra-chromosomal, and accumulates within and

outside of abnormally large ALT-associated PML bodies. I also find that when FANCD2 is

depleted there is an ALT specific increase in RAD51 colocalization with telomeric foci,

telomeric recombination reactions, telomere entanglements, and telomeres that express

characteristics of fragile sites. In two ALT cell lines FANCD2 depletion leads to microscopically

visible intra-nuclear RAD51 fibers, which may indicate a defect in the regulation of RAD51

oligomerization. However, codepletion of RAD51 with FANCD2 does not rescue the telomere

abnormalities in FANCD2 depleted cells, suggesting that the telomere phenotype is promoted by

a RAD51-independent process. Significantly, codepletion of BLM with FANCD2 completely

suppresses the telomere abnormalities in FANCD2-depleted ALT cells. I have also previously

found that FANCD2 depletion results in reduced viability in ALT cells, which codepletion of

BLM with FANCD2 partially rescues. Together, our observations provide evidence for a model

in which the FANCD2 serves to negatively regulate BLM-dependent amplification and

recombination reactions between ALT telomeric DNA.

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3.3 Materials and Methods

Cell culturing. GM00847 (GM847), Wi38-VA13/2RA (VA13), U2OS, GM00637 (GM637),

SAOS-2, GM00639 (GM639), HT1080, HeLa and PD20 cells were grown in DMEM

supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cells were mycoplasma

free. When required, cells were manually counted using a haemocytometer. Cells for colony

forming assays were replated 5 days after siRNA addition in 6 cm dishes at densities of 125,

250, 500, 1000, 10 000, or 20 000 cells per plate. 12-16 days post plating colonies were fixed in

methanol for 15 minutes, stained in 10% Giesma stain for 15 minutes, and then manually

counted.

Immunofluorescence microscopy. Cells were grown on glass coverslips, then processed for

immunostaining as previously described (Stavropoulos et al., 2002). Primary antibodies used

were rabbit anti-FANCD2 (Novus Biologicals:100-182), goat anti-TRF1 (Santa Cruz: sc-6165),

mouse anti-TRF2 (Imgenex: IMG-124); rabbit anti-PML (Millipore: AB1370); rabbit anti-

RAD51 (Merck: PC130). Mouse anti-PML 5E10 was a kind gift from Dr. Roel van Driel. All

antibodies were validated for use in IF by testing staining in cells with reduced protein levels

(patient cell lines or cells targeted with siRNA) or verifying that multiple independent antibodies

(not listed) recognized the same nuclear structures using IF.

Images were obtained using 20, 40 or 63 times 1.4NA objectives mounted onto a Zeiss Axioplan

2 microscope equipped with a Hammamatsu Orca ER camera. 12-bit grayscale images were

captured using Openlab software (Improvision). Slides from a single experiment were all

processed and imaged at the same time, using identical exposure times. For colocalization

experiments, average nucleoplasmic fluorescence was determined using Openlab, then images

were thresholded so only foci with a 2 fold intensity over background nucleoplasmic staining

were analyzed. Foci number and colocalization were manually determined by analysis of

Openlab images.

Immunoblotting. Cells were lysed in RIPA buffer (150mM NaCl, 10mM Tris pH 7.2, 5mM

EDTA, 0.1% SDS, 1.0% Triton X-100, 1.0% Na-deoxycholate, protease and phosphatase

inhibitors) and 10-20ug of lysate was run out on NuPAGE 4-12% Bis-Tris gels (Invitrogen).

Blots were probed with mouse anti-FANCD2 (sc-20022 Santa Cruz), mouse anti B-tubulin (sc-

5274 Santa Cruz), mouse anti-TRF2 (IMG-124 Imgenex), rabbit anti-TRF1 (ab1423 Abcam),

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rabbit anti-BLM (NB100-214 Novus Biologicals), or mouse anti-RAD51 (ab213 Abcam).

Secondary antibodies were labeled with HRP (Jackson ImmunoResearch) and chemiluminescent

detection using ECL was carried following manufacturers’ instructions (GE Healthcare).

siRNA. siRNA oligonucleotides were synthesized (Dharmacon) to target the following

sequences: FANCD2 (A, 5’-GGAGATTGATGGTCTACTA-3’ Zhu and Dutta 2006; B, 5’-

CCAGGAAGCAACCACTTTC-3’; C, FANCD2 siRNA (h): sc-35356 from Santa Cruz; D, 5’-

AACAGCCATGGATACACTTGA-3’ Howlett et al., 2005; Fan et al., 2009) BLM (5’-

GAGCACATCTGTAAATTAA-3’) RAD51 (5’-GAGCTTGACAAACTACTTC-3’ Ambrosini

et al, 2008) and control GL2 (5’-AACGTACGCGGAATACTTCGA-3’ Zhu and Dutta 2006).

1 x 105 cells in a single well of a 6 well plate were transfected with 100 nM siRNA unless

otherwise indicated, using Lipofectamine RNAiMax (Invitrogen) following manufacturers

instructions. For CO-FISH experiments cells were transfected once, and analyzed 56-60 hours

after siRNA addition. For all other experiments cells where subjected to a second round of

siRNA, 48 hours after the first transfection, and analyzed 5 or 8 days after the initial transfection.

FISH. Cells for FISH experiments were harvested, subjected to hypotonic swelling in 75mM

KCl (15min at 37°C), fixed in methanol/acetic acid, and dropped onto slide following standard

protocols. FISH was carried out as previously described (Zijlmans et al., 1997) with minor

modifications. Hybridization mixture containing 70% formamide, 0.5ug/ml telomere PNA probe

(Rho-(C3TA2)3), 0.5ug/ml FITC-pan-centromeric PNA probe (Tabori et al, 2006), 10mM Tris

pH 7.2, 0.1% blocking reagent (Boehringer), MgCl2 buffer (4.1mM Na2HPO4, 0.45mM citric

acid, 1mM MgCl2) was preheated for 3 min at 86°C, added to slides, covered with a coverslip,

than slides were heated for 3 min at 81°C, and left for 2 hours at room temperature prior to

washing. Slides were washed 2 x 15 min in 70% formamide, 10mM Tris pH7.2, 0.1% BSA, and

then 3 x 5 min in 100mM Tris pH 7.2, 150mM NaCl, 0.08% Tween 20.

CO-FISH. Cells used in CO-FISH experiments were grown in DMEM supplemented with

7.5uM BrdU and 2.5uM BrdC for 24 hr (847) and 20hr (HT1080) prior to harvesting, with

0.1ug/ml colcemid being added for the last 2 hours of growth. Cells were harvested for FISH as

described above. Slides were rehydrated in PBS, fixed in 4% PFA for 2 min, washed in PBS,

digested with pepsin (1mg/ml at pH 2.0) for 10 min at 37°C, washed in PBS, re-fixed in 4% PFA

for 2 min, and than washed in PBS. Slides were then incubated in Hoeschst 33258 (0.5 ug/ml in

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PBS) for 20 min, washed in PBS, and placed on a 55°C heating plate, covered with PBS and a

coverslip, then exposed for 45 min to light from an inverted 312nm UV box. The working

distance between the slide and UV box was less then 1 cm, and slides were moved every 5 min

to ensure equal damage to all regions of the slide. Slides were rinsed in PBS, digested for 12.5

min with Exonuclease III (3U/uL NEB), washed in PBS, then dehydrated and processed for

FISH as described above.

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3.4 Results

3.4.1 FANCD2-depletion results in a rapid, ALT-specific increase in telomeric DNA synthesis

I used a siRNA approach to reduce FANCD2 protein levels in ALT and telomerase positive cell

lines. Four different sequences targeting FANCD2 where tried, three of which reduced FANCD2

proteins levels to at or below residual amounts seen in patient cell lines with biallelic FANCD2

mutations (Figure 3-1). With this level of silencing, FANCD2 nuclear foci were no longer visible

by immunofluorescence. Five days after FANCD2 siRNA addition, I performed FISH and

microscopically examined telomeric DNA in interphase cells labeled with a rhodamine tagged

telomeric probe. Surprisingly, I found that a fraction of ALT cells had abnormally high amounts

of telomeric DNA present within nuclei (Figure 3-2). Increased amounts of telomeric DNA were

observed in GM847 and U2OS cells treated with FANCD2 siRNAs A-C, but not FANCD2

siRNA D, which does not significantly deplete FANCD2 protein levels or reduce ALT cell

viability (Figure 3-1; Figure 2-15). ALT cell populations normally have some variability in

telomeric DNA content, however the peak intensity of telomeric DNA foci in FANCD2-depleted

ALT cells greatly exceeded anything observed in cells treated with random siRNA or untreated

cells.

Figure 3-1. FANCD2 siRNA significantly reduces FANCD2 expression Western blot of FANCD2 protein levels in patient cells with biallelic FANCD2 mutations (PD20),

GM847 and U2OS ALT cells, and GM847 and U2OS ALT cells treated with random or FANCD2

targeting siRNAs five days post siRNA addition. FANCD2 siRNAs A-C significantly decrease FANCD2

protein levels.

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Figure 3-2 FANCD2-depleted GM847 and U2OS ALT cells have increased levels of

telomeric DNA GM847 and U2OS cells were treated with the indicated siRNA sequences, and then telomeric DNA

content was assessed 5 days post siRNA addition using a FISH probe targeting the telomeric G strand.

Scale bar is 20 µm. Images were taken at the same exposure time under 20x magnification.

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Increased telomeric DNA content was also observed in FANCD2-depleted VA13, GM637, and

SAOS-2 ALT cells (Figure 3-3, SAOS-2 data not shown). Depletion of FANCD2 in telomerase

positive cells did not result in an increase in telomeric DNA in GM639, HT1080, or HeLa

telomerase positive cells (Figure 3-3, HeLa data not shown). Our lab has previously observed

that overexpression of BLM also results in a rapid ALT-specific increase in telomeric DNA

synthesis (Stavropoulos et al., 2002), but to my knowledge FANCD2 is the first example of a

protein that when depleted gives this phenotype.

Figure 3-3. FANCD2-depleted GM637 and VA13 ALT cells but not GM639 or HT1080

telomerase positive cells, have increased levels of telomeric DNA Cells were treated with FANCD2 siRNA A, and then telomeric DNA content was assessed 5 days post

siRNA addition using a FISH probe targeting the telomeric G strand. Scale bar is 20 µm. Images of

random and FANCD2 siRNA treated cells were taken at the same exposure time under 20x magnification.

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3.4.2 FANCD2-depleted ALT cells accumulate ECTR DNA within and outside of abnormally large ALT associated PML bodies

Cells that have activated the ALT mechanism of telomere maintenance accumulate a unique

subtype of PML nuclear bodies, referred to as ALT-Associated PML Bodies (APBs). APBs are

characterized by the colocalization of telomeric DNA and telomere binding proteins with normal

PML body components (Yeager et al, 1999). APB number and size vary during the cell cycle,

with cells in late S/G2 typically accumulating larger ‘donut’ shaped bodies. ImmunoFISH

experiments show that large telomeric foci colocalize with PML and telomere binding proteins,

and that FANCD2-depleted ALT cells accumulate unusually high amounts of telomeric DNA

and telomere binding proteins within APBs (Figure 3-4 A). Examples of typical random siRNA

treated cells with large APBs are shown for comparison (Figure 3-4 A).

I next examined the frequency of GM847 nuclei containing more then one telomeric foci with a

diameter that exceeded 20 pixels (∼3 µm) in wild-type, random siRNA and FANCD2 siRNA A

treated cells. I found that FANCD2-depletion led to a 22 fold increase in the percentage of nuclei

with this phenotype 5 days post siRNA treatment relative to random siRNA treated cells (Figure

3-4 B). Telomeric DNA signals within these large foci was usually so intense that it caused

arcing of the camera signal, and signals in bodies could be observed with very short exposure

times (1-5 ms). Eight days post FANCD2 siRNA treatment, approximately 55% of GM847 ALT

cells showed abnormally large telomeric foci (Figure 3-4 B). Examination of GM847 FANCD2-

depleted pro-metaphase cells shows that these large telomeric foci have telomeric DNA signals

that are significantly more intense than signals from telomeres, and that bodies are present within

the interchromosomal space (Figure 3-4 C). This supports the idea that the bulk of the telomeric

signal in large foci is composed of extra-chromosomal material.

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A)

B)

GM847 + random siRNA Day 5

GM847 + random siRNA Day 8

GM847 + FANCD2 siRNA Day 5

GM847 + FANCD2 siRNA Day 8

GM847 + Random

siRNA

GM847 + FANCD2 siRNA A

VA13 + Random

siRNA

VA13 + FANCD2 siRNA A

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C)

Figure 3-4. FANCD2-depleted ALT cells accumulate high amounts of telomeric DNA and

telomere binding proteins within APBs A) Representative images of GM847 and VA13 ALT cells with large APBs 5 days post treatment with

random or FANCD2 siRNA A. Nuclei were stained for PML, telomeric DNA, and the telomere binding

protein TRF2. Scale bar is 5 µm. B) Plot of the number or nuclei containing more then one large

telomeric foci 5 and 8 days post treatment with random (blue) or FANCD2 siRNA A (red) treated cells

from 311 randomly selected asynchronous cells from 3 independent experiments ± the associated

standard error for each condition. C) Image of a pro-metaphase FANCD2-depleted GM847 cell 8 days

post siRNA addition. DNA is stained (blue) and telomeric DNA (red) was detected with FISH and

imaged for 200ms (left) and 5ms (right). Scale bar is 5 µm. Large telomeric foci are significantly brighter

than telomeres and accumulate in the extra-chromosomal space between chromosomes, suggesting that

the bulk of this material is ECTR DNA.

In collaboration with Andrew Larsen and Dr. Ren Li, energy-filtered transmission electron

microscopy was used to analyze APB ultrastructure. APBs in wild-type cells were shown to have

an outer protein shell (nitrogen enriched) surrounding a nuclei acid and protein core (nitrogen

and phosphorous present), and the nucleic acid within APBs differed significantly from

chromatin, most likely representing non-nucleosomal ECTR DNA (Figure 2-11). APBs in

FANCD2-depleted cells had a similar organization, but were physically larger and sometimes

contained chromatin-like material within the APB (Figure 2-12). Transcription of telomeres

results in production of a non-coding UUAGGG-repeat containing telomeric RNA, which may

have roles in chromatin remodeling or telomere capping (Azzalin et al, 2007). Telomeric RNA

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associates with telomeric DNA, and is present at higher levels in primary and ALT cells then in

telomerase positive cells (Ng et al, 2009). In order to determine whether FANCD2-depletion

leads to an increase in telomeric DNA and/or RNA, Dr. Ren Li examined the relative

phosphorous contents in serial sections of FANCD2-depleted APBs that were untreated, or

treated with RNase prior to imaging. As an internal control for the RNase treatment, sections

were chosen which contained an APB and a nucleolus. RNase treatment prior to imaging

significantly decreases the phosphorous content of the nucleolus, but does not affect the

phosphorous content of APBs, suggesting that the majority of material within FANCD2-depleted

APBs is DNA (Figure 3-5).

Bromodeoxyuridine (BrdU) is a nucleoside analog which when added to cells becomes

incorporated into DNA during replication, and can subsequently be detected with an antibody

using traditional immunofluorescence. Denaturation or cleavage of double-strand DNA that has

incorporated BrdU is required prior to immunofluorescence to make the DNA accessible to the

antibody, whereas BrdU that has been incorporated into single-strand DNA is detectable without

these treatments. I find that when GM847 and VA13 ALT cells are grown in the presence of

BrdU for 24 hours, then stained for BrdU incorporation without DNase or HCl treatment, APBs

are readily detectable, suggesting that they contain a high amount of single- strand DNA (data

not shown). Upon FANCD2 depletion, there is an increase in the size, intensity, and frequency of

APBs with detectable BrdU, suggesting that there is an increase in single-strand DNA within

APBs. In the U2OS ALT line, which normally has smaller APBs then GM847 and U2OS, BrdU

is only readily detectable in FANCD2 depleted cells (Figure 3-6 A). Supporting the idea that

APBs contain ssDNA, DAPI staining, which relies on intercalation of a fluorescent molecule

into double-strand DNA, is often poor in nuclear regions containing abnormally large telomeric

foci (Figure 3-6 B). The observation that APBs contain single-strand DNA is consistent with in

vitro analysis of ECTR DNA, and electron microscopy experiments, which suggest that the bulk

of the material within APBs differs significantly from chromatin (Nabetani and Ishikawa, 2009).

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Figure 3-5. APBs in FANCD2-depleted cells contain high levels of DNA, not RNA Serial nuclear sections containing a nucleolus (solid arrow) and an APB (dotted arrow) were imaged to

determine the nitrogen (N) and phosphorous (P) content without RNase treatment (rows 1 and 3) and with

RNase treatment prior to imaging (rows 2 and 4). A coloured overlay (O) is shown where nitrogen is blue,

and phosphorous is yellow. Scale bar of the top 2 rows is 0.5 µm. The bottom 2 rows show a higher

magnification of the APB, the scale bar is 0.2 µm. The phosphorous content of the nucleolus, but not the

APB, decreases with RNase treatment.

GM847 + FANCD2 siRNA A

GM847 + FANCD2 siRNA A + RNase

GM847 + FANCD2 siRNA A

GM847 + FANCD2 siRNA A + RNase

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A)

B)

Figure 3-6. Large APBs contain high amounts of single-strand DNA A) U2OS cells treated with FANCD2 siRNA form large telomeric foci (red) that contain single-strand

DNA (green), as assessed by the ability to detect incorporated BrdU without cleavage or denaturation of

the DNA. BrdU is not normally detectable in smaller telomeric foci in random siRNA treated U2OS cells.

Scale bar is 10 µm. B) Abnormally large telomeric foci in FANCD2-depleted cells (red, 10ms exposure)

frequently correspond to DAPI poor staining regions of the nucleus (arrows). Scale bar is 10 µm. Insert in

corner is 2.5x magnified.

DAPI DAPI + Telomeric DNA

DAPI BrdU TRF1

U2OS + FANCD2 siRNA A

U2OS + Random

siRNA

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In addition to accumulating telomeric DNA within APBs, FANCD2-depletion results in nuclei

that have an excessive number of smaller telomere signals outside of large APBs (Figure 3-7 A).

FISH using a pan-centromeric FITC labeled probe set was used in conjunction with a rhodamine

labeled telomere probe, and the frequency of telomere to centromere signals was determined.

Interphase GM847 wild-type and random siRNA treated cells have an average telomere to

centromere ratio of ∼2. FANCD2 depletion in GM847 cells can result in up to a 10-fold increase

in the telomere to centromere ratio, something that is never observed in controls. I scored the

frequency of nuclei with a telomere to centromere ratio greater then 4, and found that <1% of

random siRNA treated cells showed this phenotype, while 14% and 36% of FANCD2-depleted

cells had excess telomeric DNA signals 5 and 8 days post siRNA treatment, respectively (Figure

3-7 B). Similar increases in telomere signals were seen in U2OS ALT cells, but were not

observed in FANCD2-depleted HT1080 telomerase positive cells (0 out of 300 cells analyzed),

arguing that this is an ALT specific change. Examination of metaphase spreads shows that the

excess telomeric DNA in FANCD2-depleted cells does not accumulate at interstitial sites, but

rather is extra-chromosomal (Figure 3-7 C). Together, this argues that FANCD2-depletion

results in excess generation of ECTR DNA, which accumulates both within, and outside of

APBs.

3.4.3 FANCD2-depleted ALT cells do not upregulate expression of BLM, TRF1 or TRF2

Overexpression of BLM also results in an ALT-specific increase in telomeric DNA synthesis,

which accumulates in abnormally large foci (Stavropoulos et al., 2002), similar to what is

observed when FANCD2 is depleted. Western blot analysis of BLM expression levels in random

or FANCD2 siRNA treated cells from 4 different ALT cell lines failed to show an increase in

BLM expression (Figure 3-8), arguing that the FANCD2-depletion telomere phenotype is not an

artifact of BLM overexpression. I also examined the levels of TRF2 and TRF1, two major DNA

binding proteins that play important roles in telomere capping and replication (Takai et al, 2003;

Sfeir et al, 2009). Interestingly, I find that there is no increase in TRF2 or TRF1 expression

levels in FANCD2 depleted cells (Figure 3-8), despite the apparent increase in telomeric DNA

synthesis.

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A)

B)

C)

Figure 3-7 FANCD2-depleted ALT cells accumulate ECTR DNA outside of APBs

A) GM847 nuclei treated with random or FANCD2 siRNA 8 days post siRNA treatment. Centromeres

(green) overlayed with DAPI and telomeric DNA (red) are shown. The random siRNA treated nucleus

has a typical telomere to centromere ratio, whereas the FANCD2 depleted nucleus has a high number of

small telomeric DNA foci. Scale bar is 5 µm. Exposure times are identical. B) Plot of the number or

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nuclei containing 4x more telomeric than centromeric signals 5 and 8 days post treatment with random

(blue) or FANCD2 siRNA A (red) from 311 randomly selected asynchronous cells from 3 independent

experiments ± the associated standard error for each condition. C) Metaphase spreads of U2OS cell

treated with random (left) or FANCD2 siRNA B (right) 5 days post addition. Telomeric DNA is shown in

red. Smaller telomeric DNA signals are extra-chromosomal, not interstitial.

Interphase FANCD2-depleted ALT cells containing very large APBs often have fewer TRF1 and

TRF2 foci then would be predicted based on my FISH results, which show that 87% of FANCD2

depleted cells with large APBs have >2x more telomeric DNA signals then centromeres. This

suggests that not all telomeric DNA is detectable by TRF1 or TRF2 staining. Electron

microscopic analysis argues against APBs as being places where large numbers of telomeres are

aggregated (chapter 2), eliminating this as a possible explanation. An alternate explanation for

the low numbers of TRF1 and TRF2 foci seen in FANCD2-depleted ALT cells with large APBs,

is that there is an insufficient amount of TRF1 and TRF2 within the cell to bind to all the

telomeric DNA present within the nucleus. This could lead to telomere uncapping, and more

frequent activation of the DNA damage response by telomeric DNA with insufficient amounts of

shelterin associated proteins, explaining are previous observation that FANCD2-depletion causes

an ALT specific increase in telomere dysfunction induced foci (Figure 2-10 A).

Figure 3-8. FANCD2-depleted ALT cells do not increase expression of BLM, TRF1, or TRF2

Western blot analysis of GM847, VA13, U2OS and SAOS-2 ALT cells 5 days post siRNA treatment with

random or FANCD2 siRNA shows that BLM, TRF1, and TRF2 protein levels are not upregulated in cells

with reduced FANCD2 protein levels.

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3.4.4 FANCD2-depleted ALT cells have increased association of RAD51 with telomeric foci, telomere sister chromatid exchanges, fragile telomeres, and telomere entanglements

A key step in homologous recombination is the initial search for homology, which often involves

the assembly of a RAD51 nucleofilament on a 3’ ssDNA tail. FANCD2 is a member of a

complex of unknown function containing BRCA2, FANCG, and XRCC3 (Wilson et al, 2008).

BRCA2 plays numerous roles in controlling RAD51, including targeting RAD51 to double-

strand breaks and regulating RAD51 oligomerization (Yuan et al, 1999; Davies et al, 2001;

Pellegrini et al, 2002). FANCD2-deficient human cell lines do not show impaired RAD51 foci

formation after DNA damage (Godthelp et al, 2002; Godthelp et al, 2006), however they do have

impaired recombinational repair in reporter assay systems (Nakanishi et al, 2005).

RAD51 associates with telomeric foci and APBs at levels visible by immunofluorescence in

ALT cells, and associates with telomeric DNA transiently during the cell cycle at levels

detectable by chromatin immunoprecipitation in telomerase positive cells (Yeager et al, 1999;

Verdun and Karlseder, 2006). I therefore examined the effect of FANCD2 depletion on RAD51

colocalization with telomeric proteins in ALT and telomerase positive cells. Depletion of

FANCD2 results in 4.3 and 3.2 fold increases in the number of RAD51 foci that colocalize with

TRF2 in GM847 and VA13 ALT cells, respectively (Figure 3-9 A). In both ALT and telomerase

positive cells, FANCD2-depletion results in an increase in RAD51 foci not associated with

TRF2. However in telomerase positive GM639 there is no change in RAD51 colocalization with

TRF2, and only a 0.1 fold increase in HT1080 cells, suggesting that the increased association

between RAD51 and TRF2 is an ALT specific phenomenon.

Interestingly, when FANCD2 is depleted in U2OS and SAOS-2 ALT cells, ∼5% of cells show a

novel phenotype wherein RAD51 forms elongated structures that run through the nucleus (Figure

3-9 B). This phenotype was only rarely observed in control cells (<0.1%), and can be seen using

multiple FANCD2 siRNAs and RAD51 antibodies (data not shown). Overexpression of RAD51

can cause higher order RAD51 structures to form in some cell types, however these appear to

differ from the RAD51 fibers that I observe in FANCD2-depleted cells, which run between

distinct foci, and are not limited to non-cycling cells (Raderschall et al, 2002). Western blot

analysis does not show an increase in RAD51 expression in FANCD2-depleted cells, and

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A)

B)

C)

Figure 3-9. FANCD2 depletion effects RAD51 foci formation and oligomerization A) Plot of the number of RAD51 foci that do (red), and do not (blue) colocalize with TRF2 in populations

of 300 randomly selected asynchronous cells 5 days post treatment with random or FANCD2 siRNA A

from 3 independent experiments ± the associated standard error. FANCD2-depletion results in an increase

in non-telomeric associated foci in all cell lines tested. In ALT cells there is an increase in the number of

RAD51 foci that colocalize with TRF2, which is not observed in telomerase positive cell lines.

B) Immunofluorescence of endogenous RAD51 in FANCD2-depleted SAOS-2 cells. FANCD2 depletion

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results in formation of extended RAD51 oligomers in ∼5% of cells. Scale bar is 5 µm. C) Western blot

(left) of SAOS-2 cells treated with random siRNA, FANCD2 siRNA, or FANCD2 siRNA in conjunction

with 50nM or 100nM RAD51 siRNA. Cells depleted of RAD51 do not appear to overexpress RAD51 via

western. Immunofluorescence of RAD51 staining in SAOS-2 cells treated with FANCD2 siRNA A and

RAD51 siRNA (100nm) demonstrating that RAD51 oligomers still form and are not an artifact of

overexpression. Scale bar is 5 µm.

structures continue to form when RAD51 protein levels are partially reduced by codepleting

RAD51 with FANCD2 (Figure 3-9 C). As these structures are not observed in GM847 and VA13

ALT cells, formation of extended RAD51 structures appears to be a cell line specific, but not an

ALT specific phenomenon.

I next examined the frequency of telomere sister chromatid exchange (T-SCE) reactions in ALT

and telomerase positive cells treated with random or FANCD2 siRNA. Because telomeres have a

characteristic strand asymmetry, chromosome orientation-FISH (CO-FISH) can be used to

provide detailed information on exchanges occurring between telomeric repeat sequences.

CO-FISH involves the selective degradation of the newly replicated strand, which requires one

complete round of replication in the presence of BrdU/BrdC (Figure 3-10 A). Fanconi anaemia

cells can cycle slowly and accumulate cells with a 4N DNA content, a phenomenon which is

further heightened by treatment with DNA crosslinking agents, which results in a prolonged S

phase due to incomplete replication (Dutrillaux et al, 1982; Akkari et al, 2001).

To reduce the probability of false positives due to incomplete replication in the presence of

BrdU/BrdC, cells were labeled for 24 hr immediately after FANCD2 knockdown (36 hours after

siRNA addition), and microscopically analyzed to confirm that all cells in G2 and mitosis had

incorporated BrdU. I examined more then 3500 chromosomes over three independent

experiments, using conditions designed to ensure compete degradation of the newly synthesized

strands (45 min exposure to UV light source located <1 cm from slides place on an 55C heating

block, followed by 12.5 min digestion with ExoIII).

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B)

C)

A)

Figure 3-10. SiRNA depletion of FANCD2 in GM847 ALT cells results in increased

frequency of T-SCEs A) Overview of CO-FISH assay. Cells are replicated in the presence of BrdU/BrdC for a single round of

replication, than are harvested, and spread on slides for FISH experiments. Treatment of slides with

Hoechst, UV, and Exonuclease III results in the selective degradation of the newly synthesized. Sister

chromatid exchanges within telomeres result in a split signal on sister telomeres. B) Examples of T-SCEs

(arrows) in GM847 FANCD2-depleted cells. Centromeres are shown in green, telomeres are shown in

red. C) Plot of the average frequency of chromosome ends with evidence of a potential T-SCE event,

from 3 independent experiments ± standard deviation between experimental means. More then 2000

chromosome ends were analyzed per experiment.

GM847 GM847 + GM847+ random FANCD2 siRNA siRNA

GM847 + FANCD2 siRNA

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I find that 4.0% of GM847 ALT chromosome ends have a T-SCE, which increases slightly to

5.1% upon treatment of cells with random siRNA, and more significantly to 14.5% of

chromosome ends in FANCD2 depleted cells (Figure 3-10 C). The fraction of telomeres in a cis

pattern, indicative of a genomic SCE event, did not significantly increase in FANCD2-depleted

cells (Figure 3-14), consistent with the observation that FA proteins do not suppress genomic

SCEs in human cells (Hemphill et al, 2009).

In HT1080 telomerase positive cells I do not observe an increase in T-SCE following FANCD2

depletion, with 1.2% of HT1080 chromosome ends showing evidence of a T-SCE, 1.5% of

chromosome ends in random treated cells, and 1.4% of chromosome ends in FANCD2 depleted

cells. The frequency of T-SCEs in HT1080 cells is higher then what is normally observed in

telomerase positive, however I do not feel that it is an artifact due to incomplete incorporation or

digestion, as all cells in G2 and mitosis appeared to have incorporated BrdU, and the values

remained constant even when cells were exposed to UV for 90 minutes followed by a 20 min

digestion by ExoIII.

Increased SCEs at specific genetic loci are one feature of difficult to replicate sequences known

as fragile sites. It has recently been proposed that telomeres share characteristics with common

fragile sites, in part because when grown under conditions of replication stress they begin to have

a fragmented appearance detectable by FISH (Sfeir et al, 2009). ALT telomeres share many

characteristics with fragile sites, even when cells are not subjected to replication stress.

Examination of more then 2000 telomeres from GM847 and U2OS ALT cells, and GM639 and

HT1080 telomerase positive cells, showed that ALT telomeres frequently have a fragmented

appearance by FISH (Figure 3-11). In HT1080 and GM639 telomerase positive cells, 0.6%

(14/2337) and 0.7% (16/2183) of telomeres are fragmented, respectively. In GM847 and U2OS

ALT cells this phenotype is significantly more frequent at 7.1% (161/2256) and 25.0%

(558/2228), respectively.

HT1080 and GM847 cells were also treated with random and FANCD2 siRNA A, and examined

for changes in the frequency of telomeres with a fragmented appearance. The frequency of

fragmented telomeres in random siRNA treated HT1080s was 0.6% (13/2166). Following

FANCD2 depletion the frequency did not significantly increase at 0.7% (15/2205). This differed

from GM847 cells, which showed a 2-fold increase in fragmented telomeres after FANCD2

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depletion. 6.2% (138/2227) of GM847 cells treated with random siRNA were fragmented,

whereas 13.1% (326/2496) of FANCD2-depleted cells were fragmented.

Figure 3-11. ALT telomeres more frequently exhibit FISH staining patterns characteristic

of fragile sites then telomerase positive cells

Metaphase spread of a HT1080 telomerase positive cell (left) and a U2OS ALT cell (right) showing

telomeres (red) with abnormal staining pattern (arrows).

In addition to increases in T-SCEs and fragmented telomeres in FANCD2-depleted ALT cells, I

also observed an increase in telomeric DNA entanglements. Telomere FISH in interphase cells

occasionally revealed the presence of linear telomeric DNA fibers connecting telomeric foci

(Figure 3-12 A). These often run between larger telomeric foci, which likely represent APBs,

and smaller foci, which may represent telomeres. As cells entered prometaphase and chromatids

began to condense and move around, clear evidence of telomeric DNA connecting telomeres to

extra-chromosomal telomeric DNA foci is observed (Figure 3-12 B). This suggests that at least

some telomeres transiently associate with APBs. The frequency and extent of these telomeric

DNA bridges increases in GM847 cells when FANCD2 was depleted. At least one linear

telomeric bridge was observed in approximately 15% of wild-type and random siRNA treated

GM847, this increases to 44% and 61% in FANCD2-depleted cells, 5 and 8 days post siRNA

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addition, respectively (Figure 3-12 C). HT1080 cells only rarely showed linear telomeric fibers

connecting foci (<1%), and this value did not increase upon FANCD2 depletion.

Figure 3-12. FANCD2-depleted GM847 ALT cells frequently show evidence of telomeric

DNA entanglements A) Interphase nucleus from a FANCD2-depleted GM847 cells 5 days post siRNA addition stained for

DAPI (left) and telomeric DNA (right). Arrows indicated telomeric DNA fibers connecting foci.

B) Pro-metaphase FANCD2-depleted GM847 cell showing telomeric DNA connections running between

telomeres and large telomeric foci that likely represent APBs. Scale bars are 10 µm. C) Plot of the number

of interphase nuclei containing at least one linear telomeric DNA fiber from populations of 311 randomly

selected asynchronous cells treated with random (blue) or FANCD2 siRNA A (red) from 3 independent

experiments ± the associated standard error.

A) B)

C)

DAPI Telomeric DNA DAPI + Telomeric DNA

GM847 + FANCD2

siRNA

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3.4.5 Telomere abnormalities in FANCD2-depleted ALT cells are generated through a BLM dependent, largely RAD51 independent mechanism

Using siRNA targeting BLM and RAD51, I significantly reduced expression of BLM in

conjunction with FANCD2, or RAD51 with FANCD2 in GM847 ALT cells (Figure 3-13 A).

BLM silencing was sufficient to reduce BLM expression to the level where BLM foci were no

longer detectable by immunofluorescence, whereas only faint RAD51 foci remained detectable

in a small fraction of cells under 63x magnification (data not shown). I next examined 300 nuclei

over 3 independent experiments for the level of ECTR DNA, scoring the frequency of nuclei

with more then one abnormally large telomeric foci (>20 pixels, ∼3µm) or a telomeric to

centromeric DNA ratio greater then 4. Surprisingly, I found that codepletion of BLM with

FANCD2 completely suppressed these phenotypes, whereas codepletion of RAD51 with

FANCD2 had no effect (Figure 3-13 B, C).

Depletion of FANCD2 alone results in 35% and 55% of cells having at least one abnormally

large APBs 5 and 8 days post siRNA treatment, respectively. However when BLM is codepleted

<1% of cells show this phenotype (Figure 3-13 C). Codepletion of RAD51 with FANCD2 does

not rescue this phenotype, as 35% and 67% of cells have at least one abnormally large APB, 5

and 8 days post siRNA treatment. Likewise, 14% and 36% of FANCD2-depleted cells have a 4

fold excess of telomeric signals to centromeric signals 5 and 8 days post siRNA addition, figures

which decrease to <1% when BLM is codepleted, but remain elevated at 13% and 33% in

FANCD2 and RAD51 codepleted cells 5 and 8 days post siRNA (Figure 3-13 C). The ability of

the BLM knockdown to suppress the increased amount of ECTR DNA present in FANCD2-

depleted cells was confirmed using a FANCD2 siRNA B, and experiments were repeated and

confirmed with both FANCD2 siRNA sequences in the U2OS ALT cell line (data not shown).

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A)

B)

C)

Figure 3-13. BLM, but not RAD51, is required to generate high levels of ECTR DNA in

FANCD2-depleted ALT cells A) Western blot analysis of BLM, FANCD2, and RAD51 protein levels in GM847 cells 5 days post

siRNA addition. Both BLM and RAD51 are efficiently codepleted with FANCD2 upon siRNA addition.

B) GM847 cells treated with FANCD2 and RAD51 siRNA (left) continue to show high levels of ECTR

DNA, both within and outside of large APBs. GM847 cells treated with FANCD2 and BLM siRNA

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(right) no longer have elevated levels of ECTR DNA. Centromeric DNA (green) is overlayed with DAPI.

Telomeric DNA is shown in red. Exposure times were the same between the 2 nuclei. Scale bar is 10µm.

C) Plot of the number of nuclei with a telomeric : centromeric DNA ratio >4 (green) and the number of

nuclei with >1 telomeric foci with a diameter greater then 3µm (blue) from populations of 300 randomly

selected asynchronous GM847 cells 5 and 8 days post siRNA addition ± the associated standard error.

I next examined the effect of BLM and RAD51 codepletion on the frequency of T-SCEs in

FANCD2-depleted ALT cells. I find that codepletion of BLM, but not RAD51, is sufficient to

suppress the increase in T-SCEs observed in FANCD2-depleted cells (Figure 3-14). While

14.5% of chromosome ends in GM847 FANCD2-depleted cells have undergone a T-SCE, this

frequency declines to 6.1% when BLM is codepleted. Consistent with the established role of

BLM in suppressing genomic SCEs, the frequency of telomere G-strand signals in a cis pattern,

indicative of a single SCE event upstream of the telomeres, increases approximately 3 fold when

BLM is codepleted (Figure 3-14). Codepletion of RAD51 with FANCD2 does not decrease the

frequency of T-SCEs, which remains at 15.0%.

Figure 3-14. Codepletion of BLM with FANCD2, but not RAD51, suppresses the increase

in T-SCEs observed in FANCD2-depleted GM847 cells Plot of the average number of chromatid ends with a T-SCE (blue, left axis) in siRNA treated cells from 3

independent experiments ± the standard deviation between experimental means. More than 2000

chromatid ends were analyzed per experiment. Plot of the average number of chromosomes with a

telomere pattern in cis (orange, right axis) from 3 independent experiments ± the standard deviation

between experimental means.

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A)

B)

The increased telomere DNA entanglements observed in interphase and prometaphase FANCD2-

depleted cells also appears to be mediated in a BLM dependent manner. Although BLM and

FANCD2 depleted cells no longer form abnormally large APBs, telomeric DNA fibers can still

be occasionally observed running between larger telomeric foci and telomeres in FANCD2 and

BLM codepleted prometapshase cells (Figure 3-15 A). Codepletion of BLM with FANCD2

reduced the frequency of interphase cells with visible linear telomere fibers from 44% and 61%

in FANCD2-depleted cells, 5 and 8 days post siRNA, to 22% and 17% in FANCD2-depleted

cells when BLM is codepleted (Figure 3-15 B). Codepletion of RAD51 with FANCD2 does not

reduce the frequency of cells with telomere entanglements, which remain elevated at 50% and

60%, 5 and 8 days post siRNA addition.

Figure 3-15. Codepletion of BLM with FANCD2, but not RAD51, suppresses the increase

in telomeric DNA entanglements observed in FANCD2-depleted GM847 ALT cells A) Pro-metaphase FANCD2 and BLM depleted GM847 cell showing a telomeric DNA connection

running between a telomere and a large telomeric foci that may represent an APB (arrow). Telomeric

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DNA is shown in red, chromosomes are DAPI stained. B) Plot of the number of interphase nuclei

containing at least one linear telomeric DNA fiber from populations of 300 randomly selected

asynchronous siRNA treated cells from 3 independent experiments ± the associated standard error.

3.4.6 Codepletion of BLM with FANCD2 improves the viability of FANCD2 depleted ALT cells

I previously found that depletion of FANCD2 significantly reduces the long and short term

viability of ALT cells. As codepletion of BLM with FANCD2 rescues telomere abnormalities, I

examined whether it also rescued the loss of viability phenotype. When FANCD2 and BLM are

codepleted, the number of cells remaining 5 days post siRNA treatment is reduced relative to

controls, but increased relative to FANCD2 depleted cells alone across 4 different ALT cell lines

from 2 independent experiments. In FANCD2-depleted GM847 cells, the number of cells 5 days

post siRNA addition relative to random siRNA treated cells is 15.8%, which doubles to 32.0%

when BLM is codepleted. FANCD2-depleted U2OS show a more modest increase from 34.3%

to 49.1% when BLM is codepleted. GM637 and VA13 ALT show more striking 3 and 4 fold

increases, with relative cell numbers of 15.8% and 7.4% in FANCD2 depleted cells that increase

to 50.4% and 30.6% when BLM is codepleted, respectively.

To assess the long-term viability of remaining cells, 5 days post siRNA treatment cells were re-

plated for colony forming assays (Figure 3-16). The average colony forming ability of GM847

cells treated with FANCD2 siRNA relative to random siRNA treated cells increased from 19.2%

to 55.8% when BLM was codepleted. In U2OS cells the colony forming ability increased from

33.7% in FANCD2 depleted cells to 64.0% when BLM was codepleted. Codepletion of BLM

completely rescued the colony forming ability of FANCD2-depleted GM637 cells, which

increased from 30.3% to 105.9%. Because FANCD2-depleted VA13 cells are unable to form

colonies, this cell line was not included in these experiments.

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Figure 3-16. Codepletion of BLM with FANCD2 partially restores the colony forming

ability of ALT cells GM847 (top row), GM637 (middle row) and U2OS (bottom row) were treated with random, FANCD2, or

FANCD2 and BLM siRNA, then 500 cells were replated for colony forming assays 5 days after the initial

transfection, and stained for colonies 2 weeks after initial plating.

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3.5 Discussion

In this study I have shown that transient depletion of FANCD2 results in the rapid ALT-specific

increase in ECTR telomeric DNA content in a fraction of cells. Energy-filtered transmission

electron microscopic analysis of serial sections treated and untreated with RNase clearly

indicates that material within bodies is DNA, and not RNA, and the morphology and

phosphorous signal intensity reveals that the bulk of this material is not chromatin, but likely

ECTR DNA. One possible source of some of this ECTR DNA is that it is generated from

telomere rapid deletion events. In human cells, the 3’ssDNA telomeric overhang can invades

proximal dsDNA forming large duplex loop known as a ‘t-loop’ with a partial or full Holliday

junction at its base (Griffith et al, 1999). Expression of a mutant allele of TRF2 appears to

promote aberrant resolution of this structure, resulting in rapid shortening of telomeres and

concomitant production of circular ECTR DNA (Wang et al, 2004). Genetic analysis of this

mechanism show a dependency on NBS1, and the Rad51 paralog XRCC3, which is part of a

complex with RAD51C that is associated with Holliday junction resolvase activity (Wang et al,

2004; Liu et al, 2004). Aberrant resolution of t-loops may also contribute to production of

circular ECTR in ALT cells, as this process is also largely dependent on NBS1 and XRCC3

(Compton et al, 2007). However, I have previously observed that FANCD2 depletion does not

cause the frequency of signal free ends to increase (chapter 2), which would be expected to occur

if the bulk of the ECTR DNA was being generated through cleavage of telomeres themselves.

Experiments in different mutant backgrounds suggest that additional mechanisms can be

involved in generating circular ECTR DNA. Telomerase positive human cells deficient in WRN,

the RecQ helicase implicated in Werner syndrome, have elevated levels of circular ECTR DNA,

which is generated in an XRCC3 independent mechanism (Li et al, 2008). The KU proteins

involved in non-homologous end joining also appear to be involved in suppressing formation of

circular ECTR in Arabidopsis, in a mechanism that is independent of MRE11, the RAD51

paralogs, and the SMC6 homolog implicated in intra-chromatid recombination (Zellinger et al,

2007). Additionally, it should be noted that eukaryotic cells can also contain non-telomeric extra-

chromosomal circular DNAs, which arise from genomic elements not known to form DNA

lariate structures (Gaubatz and Flores, 1990). The mechanistic details of how non-telomeric

extra-chromosomal circular elements are generated are largely unclear, but a genetic requirement

for ligase IV in the production of extra-chromosomal circular elements involving major satellite

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DNA has been demonstrated, suggesting a mechanism where linear extra-chromosomal DNA

may also be subsequently ligated together to generate circular DNA (Cohen et al, 2006). ALT

cells contain both linear and circular ECTR DNA, raising the possibility that circular DNA can

also be generated by ligation of linear telomeric DNA substrates.

When FANCD2 is depleted, cells accumulate ECTR DNA both within and outside of APBs. One

possibility is that small pieces of ECTR outside of APBs represent a different type of DNA, such

as double-strand circular DNA, which does not activate cell cycle checkpoints and is therefore

not targeted to APBs. Telomeric DNA within APBs may primarily contain linear species of

DNA, as well as DNA containing single strand regions. Supporting this idea, BrdU incorporation

studies show that FANCD2-depleted APBs contain high amounts of ssDNA, and biochemical

purification of APBs from wild type ALT cells has shown a preferential association of linear vs

circular telomeric DNA with the bodies following DNA damage (Fasching et al, 2007).

Alternately, cells may begin to accumulate ECTR DNA outside of APBs in FANCD2 depleted

cells because it can no longer be properly targeted to APBs. Although FANCD2 depletion leads

to an increase in telomeric DNA content, expression of TRF1 and TRF2 does not appear to

upregulated. Sumoylation of telomere binding proteins appears to play an important role in

targeting telomeric DNA to APBs (Potts and Yu, 2007), raising the possibility that ECTR DNA

generated elsewhere in the nucleus can no longer be targeted to APBs in FANCD2-depleted

cells.

The production of extra-chromosomal DNA appears to be tied to genomic stability, and

accumulates at higher levels in cancer cells and cells exposed to DNA damaging agents (Cohen

et al, 1997; Cohen and Lavi, 2006; Cohen et al, 2007). Interestingly, primary FA cells, which are

inherently genomically unstable, have been reported to have both abnormally large and highly

elevated amounts of extra-chromosomal circular DNAs detectable by electron microscopy and

southern blotting of 2D gels with a Cot-1 DNA probe, suggesting that the FA pathway plays a

general role in suppressing formation of these molecules (Motejlek et al, 1993; Cohen et al,

2007). The sequences of extra-chromosomal DNA amplified in primary FA cells have not been

determined, however our FISH analysis suggests amplification of telomeric DNA is largely an

ALT-specific phenomenon, as it was not observed in FANCD2-depleted telomerase positive

cells. Whether additional sequences are amplified in FANCD2-depleted ALT cells remains to be

determined, as does the relative abundance of linear and circular ECTR DNA and the molecular

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mechanism of its production. One intriguing possibility is that non-telomeric extra-chromosomal

sequences are also amplified in FANCD2-depleted ALT cells and are also sequestered within

APBs. In addition to telomeric instability, activation of ALT is also associated with instability of

the minisatellite MS32 (Jeyapalan et al, 2005), whether or not this sequence is also present as an

extra-chromosomal element in ALT cells is presently unknown. If APBs contain multiple types

of extra-chromosomal DNA, non-telomeric sequences may occasionally interact with

dysfunctional telomeres. This would result in telomeres that contain non-TTAGGG repeat

sequences, which could lead to telomere capping abnormalities. Integration of non-telomeric

DNA into telomeres has been reported in an ALT SV40 immortalized cell line derived from a

patient with Werner syndrome, wherein SV40 DNA sequences are interspersed in TTAGGG

repeats (Marciniak et al, 2005).

FANCD2-depletion results in a very rapid accumulation of ECTR DNA within cells, suggesting

that there is an amplification step occurring, which may be driven by replication and

recombination among pre-existing ECTR DNA. ECTR DNA synthesis in FANCD2-depleted

cells is not caused by overexpression of BLM, but rather appears to be due to dysregulation of a

process involving BLM. Although telomeres are a repetitive sequence element, pre-replicative

complexes can assemble on telomeric DNA and be converted into functional origins in Xenopus

extract systems, and replication forks originating within telomeres have been observed in mice,

suggesting that replication events can arise within telomeric sequences (Kurth and Gautier, 2009;

Sfeir et al, 2009). Alternately, replication may begin without a functional origin, as has been

proposed for break induced replication in yeast. BrdU incorporation experiments have previously

shown that newly replicated DNA accumulates within APBs during a time when normal S phase

replication at other loci is not occurring (Nabetani et al, 2004). Whether or not this DNA is

synthesized within APBs or is targeted to bodies after being replicated elsewhere is presently

unclear, however, APBs have been shown to contain proteins implicated in replication including

RPA and PCNA (Grudic et al, 2007; Jiang et al, 2009).

How FANCD2 may act to limit amplification of telomeric DNA remains an open, but intriguing

question. Purified human FANCD2 not only binds to ssDNA and dsDNA, but also to 3 and 4

way DNA junctions (Roques et al, 2009). One possible function of FANCD2 within ALT would

be to limit recombination between telomeric DNAs by affecting the stability of recombinational

intermediates. Type II recombinational telomere maintenance in yeast appears to rely on Break

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Induced Replication (BIR), a mechanism in which one side of a double-strand break invades a

homologous sequence resulting in a displacement loop, which can be migrated as the invading

strand is extended, with newly synthesized single- strand being displaced and subsequently

converted to a double-strand product. Alternately, a uni-directional replication fork may

assemble within the displacement loop, allowing replication to continue until the end of the

template is reached. This may occur by a mechanism involving migration of the Holliday

junction and displacement of the newly synthesized DNA, or the Holliday junction may remain

static and have to be resolved after replication is finished (reviewed in McEachern and Haber,

2006).

Both telomeres, and linear ECTR DNA naturally represent one-sided breaks, making BIR a

likely pathway for telomere extension and ECTR DNA amplification. If strand invasion occurred

into a circular template, rolling circle replication could follow, leading to a very rapid increase in

telomeric DNA content. BIR pathways can occur through RAD51 dependent and independent

mechanisms. Genetic studies using telomerase deficient yeast relying on type II telomeric

recombination, which closely resembles ALT telomere maintenance, suggest that telomeres are

maintained through RAD51-independent form of BIR, which differs slightly from RAD51-

independent BIR at an induced double-strand break in that there is unique requirement for the

Sgs1 RecQ helicase at telomeric BIR (Signon et al, 2001; Huang et al, 2001).

Amplification of ECTR DNA in FANCD2 depleted cells also appears to occur through a largely

RAD51 independent, but BLM dependent mechanism, as codepletion of BLM, but not RAD51,

suppresses this phenotype. However, it should be noted that RAD51 is a common component of

APBs, and I observe an increase in the fraction of telomeric foci that colocalize with RAD51

when FANCD2 is depleted. This suggests that multiple mechanisms of recombination may be

occurring among human telomeric DNAs, and that RAD51 may participate in recombination

reactions when present, but additional mechanisms may function when RAD51 levels are

depleted. Support for the existence of RAD51-independent mechanisms in mammalian ALT

telomere maintenance is provided by studies in mice which show that RAD54, a RAD51

accessory factor required in RAD51-dependent telomere maintenance in yeast, is dispensable for

ALT telomere maintenance in telomerase deficient mouse cells (Akiyama et al, 2006; Chen et al,

2001). One RAD51-independent mechanism of recombination which may function in

mammalian cells may rely on TRF2, a protein present at high levels on telomeric DNA that can

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promote strand invasion and subsequent t-loop formation in vitro (Griffith et al, 1999).

Alternately, interactions between complimentary regions of single-strand telomeric DNAs may

be promoted by strand annealing pathways, by-passing the need for a recombinase.

Although BLM possesses activities that aid in suppressing recombination and sister chromatid

exchanges at a global level, it also has multiple pro-recombinogenic activities that may be

essential in promoting BIR at telomeres. BLM may be required to resolve formation of

G-quadruplex structures, which may form on the telomeric G strand prior to strand invasion.

Additionally, a role for BLM in stimulating the resection of double-strand breaks required to

generate the 3’ssDNA overhang involved in subsequent recombination reactions has recently

been demonstrated in both in vitro and in vivo systems (Nimonkar et al, 2008; Gravel et al,

2008). BLM also promotes the annealing of complimentary ssDNA regions in vitro, which may

play a role in promoting interactions between telomeric DNAs (Cheok et al, 2005). When BLM

is depleted these events may occur less efficiently, leading to a decrease in accessible ssDNA or

telomeric recombinational intermediate structures, which may explain our previous observation

that siRNA knockdown of BLM results in a significant reduction in FANCD2 localization to

telomeric foci (chapter 2). Conversely, when BLM is overexpressed there may be an increase in

interactions between telomeric DNAs and BIR, resulting in the dramatic ALT specific increase

in telomeric DNA that has been previously observed (Stavropoulos et al, 2002).

In addition to early roles in the recombinational process, BLM also possesses activities that may

be important in promoting efficient replication of telomeric DNA, including the ability to

promote branch migration of Holliday junctions (Karow et al, 2000). Additionally, both BIR and

types I and II telomere maintenance in yeast rely on pol 32, a subunit of pol δ, suggesting that

pol δ is the polymerase involved in recombinational telomere maintenance (Lydeard et al,

2007). BLM directly interacts with the p12 subunit of human pol δ, and can promote the strand

displacement activity of pol δ in vitro, which may be important for efficient replication of

gapped telomeric substrates (Selak et al, 2008).

While BLM may function in multiple ways to promote ECTR DNA synthesis, at least one of

these functions relies on the DNA unwinding ability of BLM, as overexpression of BLM with a

nonfunctional helicase domain does not result in increased telomeric DNA synthesis

(Stavropoulos et al, 2002). It is also interesting to note that the WRN RecQ helicase also

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localizes to APBs and telomeric foci in ALT cells, and similar to BLM, WRN interacts with

TRF2 in vitro and in vivo, and TRF2 stimulates the helicase activity of both BLM and WRN

(Johnson et al, 2001; Opresko et al, 2002). Both BLM and WRN can readily unwind

G-quadruplex DNA structures, yet each helicase appears to have non-overlapping roles at

telomeres (Mohaghegh et al, 2001). Codepletion of BLM with FANCD2 completely suppresses

the FANCD2-depletion telomere phenotype, suggesting that WRN is not able to substitute for

BLM. Likewise, mouse cells deficient for BLM show a higher frequency of telomeres with a

discontinuous staining pattern observed by FISH, suggested to be due to problems with telomeric

replication caused by G-quadruplexes, which is not observed in WRN deficient mouse cells

(Sfeir et al, 2009). However, in human cells, WRN deficiency appears to result in specific

problems with replication of the telomeric G strand, again suggesting that BLM cannot substitute

for the WRN helicase (Crabbe et al, 2004).

Depletion of FANCD2 in ALT cells also results in an increase in the frequency of telomeres with

a discontinuous staining pattern via FISH, which is not observed in FANCD2-depleted

telomerase positive cells. It has been suggested that this altered staining pattern is similar to what

occurs at aphidicolin induced common fragile sites, and that telomeres with this staining pattern

represent telomeres expressing fragile site characteristics (Sfeir et al, 2009). Interestingly, the

frequency of telomeres with this phenotype is increased >10 fold in the two ALT cell lines I

examined relative to two telomerase positive cell lines. It has been suggested that insufficient

expression of shelterin components may contribute to telomere abnormalities in ALT cells,

resulting in a partial capping defect (Cesare et al, 2009). In non-ALT cells, depletion of TRF1

increases both the frequency of telomeres with this fragile appearance and replication fork

stalling within telomeric DNA, suggesting that TRF1 has a role in promoting telomeric

replication and suppressing fragile telomeres (Sfeir et al, 2009). Insufficient amounts of TRF1

within ALT cells is one potential cause of the increase in fragile telomeres. While FANCD2-

depleted ALT cells have increased amounts of telomeric DNA, I do not observe a concomitant

increase in TRF1 or TRF2 expression. This further skewing of the ratio of telomeric DNA to

telomere binding proteins may be a contributing factor to both the ALT specific increase in

telomeric DNA that has activated a DNA damage response (chapter 2) and telomeres with a

fragile appearance.

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When FANCD2 is depleted I observe an increase in the frequency of T-SCEs in ALT, but not in

telomerase positive cells. T-SCEs were measured during the first round of replication after

FANCD2 protein levels are reduced, which proceeds the accumulation of high amounts of ECTR

DNA, arguing against a dramatic reduction in telomere capping proteins as a contributing factor

to this increase in T-SCEs. This suggests that FANCD2 is acting to directly limit recombination

between telomeric DNA. The observation that FANCD2 depletion results in an ALT-specific

increase in T-SCEs is at odds with a recent publication by Fan and colleagues, who observed an

apparent ALT-specific decrease in T-SCEs when FANCD2 is depleted (Fan et al, 2009). The

reason for this disparity is unclear, however the baseline level of T-SCEs observed in control

cells by Fan et al, was abnormally high at 40-45 T-SCEs per 100 chromosomes, meaning that 40-

45% of chromosomes have a T-SCE. The upper range of T-SCEs previously reported in these

cell lines is 66–105 T-SCEs per 100 metaphase spreads, which given that these cell lines have

hyperdiploid and hypertriploid chromosome numbers, corresponds to approximately 1% of

chromosomes (Londono-Vallejo et al, 2004). While there is a growing amount of variability of

reported T-SCE frequencies between groups, which may relate to FISH sensitivity, differences in

how well the newly synthesized strand is degraded, and genetic variability between cell lines, a

frequency of 40-45% of chromosome having a T-SCEs is abnormally high. Based on the

indicated BrdU labeling time used by Fan et al, elevated T-SCEs are likely due to incomplete

degradation of the newly synthesized strand. However, why levels of T-SCEs decrease when

FANCD2 is depleted is unknown, but is unlikely to be due to be superior silencing of FANCD2,

as Fan et al used FANCD2 siRNA sequence D to knockdown FANCD2, which does not

significantly reduce FANCD2 protein level (Figure 3-1 A). Interestingly, although I do not see

an increase in T-SCEs in the telomerase positive HT0180 cell line when FANCD2 is depleted,

mouse cells that have short telomeres and are deficient in both FANCC and telomerase have

elevated levels of T-SCEs, supporting a role for the FA pathway in limiting telomeric

recombination (Rhee et al, 2010).

The underlying mechanism of T-SCEs is currently unknown, but appears to differ from DNA

damage induced SCEs and spontaneous SCEs at other genomic loci. Unlike Bloom syndrome

cells that have increased spontaneous genomic SCEs, human FA cells have normal levels of

spontaneous genomic SCEs (Hemphill et al, 2009). Additionally, ALT cells deficient in RAD54

have suppressed levels of mitomycin C induced SCEs, but continue to have elevated levels of

141

T-SCEs (Akiyama et al, 2006). Whether T-SCEs in ALT cells occur through the same

mechanism as T-SCEs in non-ALT cells is presently unclear. In a non-ALT setting, T-SCEs are

likely driven by short telomeres, however elongating short telomeres in ALT cells by expressing

telomerase does not suppress elevated levels of T-SCEs (Morrish and Greider, 2009; Londoño-

Vallejo et al, 2004). Proteins implicated in T-SCE formation such as Mus81 and FANCD2

appear to affect the frequencies of T-SCEs in ALT cells, but not telomerase positive cells, which

may be due to a difference in the integrity of the telomere capping structure, or may be indicative

of a different mechanism driving the T-SCEs in the ALT setting (Zeng et al, 2009).

Codepletion of BLM, but not RAD51, reduces the increased frequency of T-SCEs observed in

FANCD2-depleted ALT cells to wild-type levels. This suggests that FANCD2 suppresses

T-SCEs that are driven by a RAD51-independent, but BLM-dependent process. The frequency of

genomic SCEs appears to increase in cells codepleted of FANCD2 and BLM, while T-SCE

frequency decreases, again suggesting a different mechanism of genomic SCE production from

T-SCEs. As discussed previously, BLM may be required to generate T-SCEs by promoting end

resection, resolving secondary structures, or promoting strand annealing. Interestingly, BLM

codepletion with FANCD2 does not completely suppress T-SCEs, but rather returns them to

wild-type levels. This may be due to residual BLM protein levels expressed in siRNA treated

cells, or may be because T-SCEs can arise from multiple mechanisms.

FANCD2 has been shown to accumulate preferentially on ssDNA in vitro, and FANCD2 foci

form at in vivo regions enriched in ssDNA (Roques et al, 2009). The function of FANCD2 at

ssDNA is presently unknown, however we observe that when FANCD2 is depleted, cells

accumulate APBs with highly elevated amounts of ssDNA, which may be a byproduct of BIR, or

an indication of excessive resection of dsDNA. FANCD2 may help to suppress telomeric

recombination reactions by binding to existing ssDNA regions, which are present at elevated

frequency within ALT telomeric DNA, and limiting their further resection, as more extended

regions of ssDNA may be involved in recombinational reactions.

When FANCD2 is depleted, cells also display more frequent entanglements involving telomeric

DNA, observed as linear telomeric DNA fibers running between telomeric foci in interphase

cells. Examination of prometaphase cells suggests that these fibers frequently involve telomeric

DNA connecting telomeres and APBs. Fibers may be formed if telomeres move away from

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APBs before replication is complete, Holliday junctions are resolved, or DNA is decatinated.

Increased frequency of telomere fibers in FANCD2 depleted cells may reflect a direct role for

FANCD2 in these processes, or may be a secondary effect of increased interactions among

telomeric DNAs. The observation that codepletion of BLM with FANCD2 returns the frequency

of telomere fibers returns to baseline levels, but does not complete suppress their formation,

suggests either that sufficient BLM remains to promote entanglements, or that entanglements

may arise through because of multiple factors, some of which are BLM independent.

The fission yeast Rqh1 RecQ helicase also promotes telomeric DNA entanglements, hyper-

recombination, and chromosome missegregation when the Taz1 telomere binding protein is

deleted, and cells are grown at lower temperatures (Miller and Cooper, 2003; Rog et al., 2009).

In this situation, telomere problems arise during replication, suggesting that they may be caused

during the restart of stalled or collapsed replication forks. The FA pathway has also been

implicated in the response to stalled or collapsed replication forks, raising the question of

whether some of the telomere abnormalities observed in FANCD2-depleted ALT cells may arise

during replication. Supporting this idea, I observe an increase in the fraction of the cell

population that shows abnormally high levels of ECTR DNA as the time post knockdown

increases, which may be accounted for if a fraction of cells encounter problems each time they

try to replicate. However, the role of the FANCD2 in the response to stalled/collapsed replication

forks appears to be regulated by ATR (Andreassen et al, 2004), and I do not see a requirement

for ATR expression for the majority of FANCD2 localization to telomeric foci (chapter 2).

Additionally, when I significantly reduced ATR expression in ALT cells, I did not observe any

increase in telomeric DNA content (data not shown), and treatment of ALT cells with caffeine to

inhibit of ATM and ATR has been reported to cause a decrease in telomeric DNA synthesis at

APBs (Nabetani et al, 2004). Therefore I presently favour the hypothesis that telomere

abnormalities in FANCD2-depleted cells arise due to a deregulation of recombination reactions

among telomeric DNA at sites unrelated to collapsed replication forks. Support for a role of

FANCD2 in regulating recombination outside of the context of replication problems is provided

by the observation that it FANCD2 is found at both telomeres and recombinational nodules

during meiosis, and FANCD2 null cells show an increase in both unpaired and mispaired meiotic

chromosomes (Garcia-Higuera et al, 2001; Houghtaling et al, 2003).

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In this study I provide evidence suggesting that FANCD2 functions to suppress recombination

reactions among telomeric DNA within the ALT telomere setting. Interactions between telomeric

DNA appear to form in a BLM-dependent, RAD51-independent process, and can become

dysregulated through overexpression of BLM, or depletion of FANCD2. While telomeric

recombination is essential for ALT telomere maintenance, if it occurs in an unregulated manner

it may lead to excessive production of ECTR DNA, telomeric DNA entanglements, and

increased levels of telomeric DNA which activates a DNA damage response. Excessive

recombination of telomeric DNA appears to lead to reduced cell viability, as ALT cells with high

levels of BLM, or low levels of FANCD2, show increased cell death. While impaired viability

may also be due to non-telomeric problems, the observation that codepletion of FANCD2 and

BLM corrects telomeric abnormalities and partially rescues viability suggest that telomeric

abnormalities are a contributing factor. This work serves to emphasize that RecQ helicases can

act as double-edged swords, acting to both promote and impair genomic stability depending on

the context. It also emphasizes the role of FANCD2 in regulating recombination, and suggests a

novel relationship between FANCD2 and BLM, wherein a function of FANCD2 is to regulate

BLM dependent processes.

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Chapter 4

4 Summary and Future Directions

4.1 Summary and Future Directions

In this thesis I provide evidence for a critical role of the FA pathway in ALT telomere

maintenance in human cells. Examination of the localization of endogenous proteins using

indirect immunofluorescence clearly shows that components of the FA core complex, as well as

FANCD2, frequently localize to telomeric foci in ALT cells, but not in telomerase positive or

primary cells. The FA core complex most likely localizes to telomeric foci to promote FANCD2

monoubiquitination, a requirement for its accumulation in ALT telomeric foci.

Coimmunoprecipitation experiments in late S/G2 cells support a specific role for FANCD2 in the

ALT pathway of telomere maintenance, as interactions between endogenous FANCD2, TRF2

and BLM were detected in ALT cells, but not telomerase positive cells.

Most colocalization events between FA and telomeric proteins occur within larger telomeric foci,

corresponding to APBs. Analysis of APBs using energy-filtered transmission electron

microscopy shows that APBs are composed of an outer protein shell surrounding an inner DNA

and protein core, and that DNA within the body differs significantly from chromatin and likely

represents non-nucleosomal ECTR DNA. FISH analysis of pro-metaphase and metaphase cells

adds support to the idea that large telomeric foci are primarily composed of ECTR DNA. This

suggests that at least one function of the FA pathway within ALT telomere maintenance involves

ECTR DNA. SiRNA depletion of FANCD2 confirms an ALT specific role for FANCD2 in the

regulation of ECTR DNA production, as a proportion of FANCD2-depleted ALT cells have

markedly elevated amounts of ECTR DNA, which accumulates both within, and outside of

APBs. The mechanism by which FANCD2 depletion causes ECTR DNA amplification is

presently unclear, but is unlikely to be solely a result of cleavage of telomeric DNA from

telomeres themselves, as FANCD2 depletion does not cause an increase in telomere signal free

ends.

The function of FANCD2 within ALT appears to be closely intertwined with that of the BLM

helicase. This is supported by the observations that 1) FANCD2 coimmunoprecipitates with

BLM in late S/G2 ALT cells, 2) FANCD2 almost always colocalizes to telomeric foci that also

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contain BLM, 3) depletion of BLM suppresses the localization of FANCD2 to telomeric foci, 4)

overexpression of BLM causes a rapid ALT specific increase in telomeric DNA similar to what

is observed in FANCD2-depleted cells (Stavropoulos et al., 2002), and 5) codepletion of BLM

with FANCD2 suppresses the increase in ECTR DNA, T-SCEs, and telomere entanglements

normally observed in FANCD2-depleted cells. Expression of BLM is not increased in FANCD2-

depleted ALT cells, ruling this out as a cause of ECTR DNA amplification. BLM may have

multiple critical functions at ALT telomeres including, but not limited to, generation of a 3’

ssDNA overhang if recombination is initiated at internal telomeric sites, resolving secondary

DNA structures to allow recombination and/or replication to occur, and/or promoting Holliday

junction migration if replication involves a break induced replication mechanism. Conversely,

there are multiple steps where FANCD2 may be acting within ALT. FANCD2 may bind to the

ssDNA overhang, blocking the invasion step in telomeric recombination, may act downstream of

this step to destabilize recombinants once they have formed, or may in some other manner

impede the replication step.

Identifying the polymerase involved in the amplification of ECTR DNA in ALT cells will allow

the potential role of FANCD2 in telomeric replication to be explored. The FA pathway has been

implicated in translesion synthesis, a process that relies on a specialized group of error prone

DNA polymerases referred to as translesion DNA polymerases. In vitro experiments demonstrate

a unique role for the human polη translesion polymerase in promoting DNA synthesis from a

displacement-loop recombinational intermediate, making this a potential candidate involved in

the amplification of ALT telomeric DNA (McIlwraith et al, 2005). However, work done in yeast

suggests that the core replicative polymerase in this process may be DNA polymerase δ.

Telomerase deficient S. cerevisiae that have escaped senescence and activated a recombination

dependent telomere maintenance pathway require expression of pol32, a subunit of polymerase δ

(Lydeard et al, 2007). Pol 32 is also required for break induced replication, but is not essential

for replication of the genome, or for gene conversion at induced double-strand breaks (Lydeard

et al, 2007). Mammalian polymerase delta consists of 4 subunits, p125, p50, p66 and p12 (Liu et

al, 2000). Intriguingly, the p12 subunit interacts with BLM both in vivo and in vitro, and the p12

subunit stimulates BLM helicase activity in vitro (Selak et al, 2009). As the production of ECTR

in FANCD2-depleted ALT cells is dependent on BLM, the p12 subunit of polymerase δ should

also be examined. A siRNA codepletion approach targeting p12 or polη, with FANCD2 could be

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performed, to determine if amplification of ECTR DNA is dependent on either of these

polymerases. Once the polymerase involved in amplification is identified,

coimmunoprecipitation and fluorescence resonance energy transfer experiments can be

performed to test for in vivo interactions between FANCD2 and the polymerase.

FANCD2 depletion results in increases in T-SCEs, fragile telomeres, telomeric DNA

entanglements, telomere dysfunction induced foci, and localization of RAD51 to telomeric foci.

These effects are all observed within FANCD2-depleted ALT cells, but not FANCD2-depleted

telomerase positive cells. Some of these effects may be secondary to telomere uncapping

problems resulting from amplification of telomeric DNA without increases in shelterin

components, however the T-SCE phenotype is seen at time points preceding visible

amplification of ECTR. To test whether the other ALT specific telomere abnormalities are also

independent of the amplification of ECTR DNA, these phenotypes should be reexamined at an

early time points following FANCD2 depletion.

The observed increase in T-SCEs in FANCD2-depleted ALT cells suggests that FANCD2 has a

direct role in either limiting recombination events between telomeres, or determining whether

recombination events result in an SCE. If FANCD2 acts to limit recombinant events between

sister telomeres, it may be playing a similar role amongst ECTR DNA, helping to prevent

excessive recombination. When FANCD2 is depleted, or BLM is overexpressed, elevated rates

of a break induced replication type mechanism between ECTR DNAs may result in excessive

amplification of ECTR DNA molecules. If amplification of ECTR DNA within FANCD2-

depleted ALT cells occurs via a break induced replication type mechanism, the recombinational

aspect of this process appears to be able to function via RAD51-independent pathways, because

codepletion of RAD51 with FANCD2 does not suppress amplification of ECTR DNA. A similar

RAD51- independent mechanism of break induced replication has been proposed to function in

telomerase deficient yeast type II survivors, which also maintain telomeres using recombination

and have heterogeneous and hypervariable telomere repeat tracts (McEachern and Haber, 2006).

FANCD2 may act to limit recombination in a number of different ways. FANCD2 has been

reported to preferentially accumulate on single-stranded DNA in vitro, and FANCD2 colocalizes

with ssDNA and RPA in vivo (Roques et al, 2009). Telomeric DNA in ALT cells contains

frequent internal ssDNA regions, which FANCD2 may localize to (Nabetani and Ishikawa,

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2009). One possible role of FANCD2 at these sites would be limit the further expansion of these

regions, potentially decreasing the frequency of recombination reactions involving internal

gapped regions. If FANCD2 acts to normally limit resection of dsDNA, this may be one

contributing factor to the increased amounts of ssDNA observed in FANCD2-depleted APBs. To

test this hypothesis experiments could be done looking at the effect that codepletion of FANCD2

with EXO1, a protein implicated in the resection of dsDNA, has on the frequency of T-SCEs

and ECTR DNA production.

Recombination between telomeric DNA could also occur during the restart of collapsed

replication forks. Treatment of cells with agents that induce replication fork collapse are potent

activators of FANCD2 monoubiquitination and foci formation (Andreassen et al, 2004; Howlett

et al, 2005). However, FANCD2 monoubiquitination in response to collapsed replication forks

appears to be an ATR dependent process, while localization of FANCD2 to ALT telomeric foci

is largely independent of ATR expression (Andreassen et al, 2004). Additionally, inhibition of

ATM and ATR causes an apparent decrease, not an increase in telomeric DNA synthesis

(Nabetani et al, 2004). Furthermore, collapsed replication forks trigger a DNA damage response,

and FANCD2 primarily localizes to telomeric foci that do not contain 53BP1.

Although T-SCEs may occur independently of APBs, FISH and electron microscopic imaging

experiments suggest that telomeres can localize to APBs. Interphase nuclei with linear telomeric

DNA fibers that connect larger telomeric foci, likely representing APBs, to smaller telomeric

foci are observed in FANCD2-depleted and control ALT cells. In FANCD2-depleted cells,

examples of prometaphase cells with telomeres connected by linear telomeric DNA fibers to

large extra-chromosomal telomeric foci are observed, consistent with a telomere moving away

from an APB with some form of unresolved replication or recombination intermediate. Electron

spectroscopic imaging of APBs in FANCD2-depleted ALT cells also shows chromatin-based

structures, which may represent telomeres, invading the bodies. Detection of APBs with

chromatin like structures within APBs in FANCD2-depleted ALT cells, but not controls, may be

an indicative of a change in the frequency or stability of such events when FANCD2 is depleted.

One major function of APBs may be to serve as a site where DNA that has already activated, or

may be prone to activating a DNA damage response is sequestered. This could include both

telomeres and ECTR DNA. Supporting this idea, large APBs contain single-stranded DNA that

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is clearly visible by immunofluorescent analysis of incorporated BrdU. To explore whether

APBs serve as a site where the DNA damage response is actively downregulated, future

experiments examining whether phosphatases and deubiquitinases also localize to APBs could

be performed. Initial enzymes that could be examined include PP2A, PP4, PP6, and USP1.

Alternately, APBs may provide a local environment that is noncondusive to activation of cell

cycle checkpoints. Targeting of telomeric DNA to APBs has been proposed to occur through the

sumoylation of telomere binding proteins by the SMC5/6 complex (Potts and Yu, 2007).

Additionally PML isoform 3 (PML3) interacts with TRF1 and may be involved in the

recruitment of telomeric DNA to APBs (Yu et al, 2009). If activation of cell cycle checkpoints is

normally impaired through the localization of telomeric DNA to APBs, then disruption of this

process through the siRNA depletion of SMC5/6 or PML3 would be predicted to cause an

immediate increase in CHK1 and/or CHK2 phosphorylation detectable by western blot analysis

or immunofluorescence with phosphorylation specific antibodies. Immunofluorescent analysis of

phosphorylated CHK1 and CHK2 expression could also be used to determine if cells with APBs

that have activated a DNA damage response, have also transiently activated cell cycle

checkpoints.

FANCD2-depleted ALT cells have an elevated amount of ECTR that accumulates outside of

APBs. One possibility is that this DNA is unable to be properly targeted to APBs due to

insufficient amounts of telomere binding proteins, and would support a model where ECTR is

generated outside of APBs, then subsequently localized to APBs. Alternately, this ECTR DNA

may primarily be circular DNA that does not activate DNA damage or cell cycle checkpoint

responses, and therefore does not present a hazard to cells. A simple experiment that could be

performed to determine on a single cell level whether or not this DNA is linear or circular would

be to try to end-label the ECTR DNA with fluorescently tagged nucleotides. Terminal transferase

can end-label non-ALT telomeres during the G2 phase of the cell cycle, and this technique can

easily be combined with immunofluorescence and FISH (Verdun et al, 2005). Using this

approach, the question of whether or not APBs contain linear ECTR DNA molecules can also be

directly addressed at a single cell level in an in vivo situation. An additional experiment that

could be used verify the results of this experiment would be to isolate ECTR DNA using a HIRT

lysate protocol, and then examine it using electron microscopy. This would not only allow for

detailed analysis of differences in the size distribution of extra-chromosomal molecules in

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FANCD2-depleted versus control cells, but also the detection of changes in the proportion of

intermediate molecules such as ongoing rolling circle replication and Holliday junction

structures.

Although APBs are assumed to only contain telomeric DNA, this assumption does not appear to

have been formally tested. Abnormally large PML bodies that appear to contain DNA within the

interior have also been described in cells from patients with immunodeficiency, centromeric

instability, and facial dysmorphy (ICF) syndrome (Luciani et al, 2006). ICF syndrome can be

caused by mutations in the DNMT3B methyltransferase which leads to hypomethylation and

instability of some GC rich regions (Hansen et al, 1999; Xu et al, 1999). These large PML bodies

appear remarkably similar to APBs when examined with light microscopy, and also accumulate

during the G2 phase of the cell cycle. However, in ICF syndrome cells, 1qh, 16qh, 9qh, and 15ph

satellite DNA, but not telomeric DNA, appears to accumulate within PML bodies (Luciani et al,

2006). Luciani and colleagues hypothesized that the DNA within PML bodies corresponds to

interchromosomal heterochromatic regions because chromosomal loci near the satellite DNA

locations are frequently located in close proximity to the PML bodies, and heterochromatic

protein 1 (HP1) colocalizes with the PML bodies. However, other heterochromatin markers

including histone 3 trimethylated on lysine 9, histone 4 trimethylated on lysine 20, and the

macroH2A variant do not colocalize with the PML bodies (Luciani et al, 2006). An alternate

hypothesis is that the bulk of the satellite DNA localized to PML bodies in ICF cells represents

extra-chromosomal material, and similar to APBs, the chromosomal regions that this material is

derived from may transiently localize to PML bodies.

The underlying similarity between ALT telomeres and ICF satellite DNAs may be that they both

represent genomically unstable regions that may be more prone to generate extra-chromosomal

DNA. APBs and the large PML bodies in ICF cells may represent a general subtype of PML

body that is involved in sequestering extra-chromosomal material. Electron spectroscopic

imaging experiments of large PML bodies in ICF cells would be an important first step in

characterizing the material within ICF associated PML bodies, and determining if similar to

APBs, this material does not correspond to chromatin. A further test of the hypothesis that PML

bodies have a generic role in sequestering extra-chromosomal material would be to isolate ECTR

DNA from an ALT cell using a HIRT lysate protocol, and transfect this material into non-ALT

cells, then examine cells for formation of APBs.

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An additional important experiment in ICF cells would be to examine the effect of FANCD2-

depletion on microsatellite DNA that has been shown to associate with PML bodies. If these

satellite sequences also become highly amplified upon FANCD2 depletion, this would suggest

that what is observed in FANCD2-depleted ALT cells is part of a wider phenomenon affecting

genomically unstable regions. A general role for the FA pathway in suppressing formation of

extra-chromosomal material is supported by studies in primary FA cells, which show the

presence of abnormally large and highly elevated amounts of extra-chromosomal circular DNAs

detectable by electron microscopy and southern blotting of 2D gels with a Cot-1 DNA probe

(Motejlek et al, 1993; Cohen et al, 2007). Although the mechanism of generating extra-

chromosomal circular DNA from repeat sequences is unknown, it can occur independently of

genomic replication, and is believed to rely on illegitimate recombination events as an initial first

step (Cohen and Mechali, 2001; Cohen et al, 2006). FANCD2 may play a critical role in these

situations by helping to limit initial recombination events.

Within ALT cells there is a report of at least one other genomic loci, the minisatellite MS32, that

shows highly elevated levels of genomic instability in a majority of ALT cells, but not

telomerase positive cells (Jeyapalan et al, 2005). An important follow up experiment to this

observation, would involve FISH analysis of this minisatellite to determine whether it also

becomes amplified as an extra-chromosomal element and localizes to PML bodies in wild-type

and FANCD2-depleted ALT cells. An additional sequence that would be important to examine

using this approach is the FRA16D fragile site locus. FANCD2 is frequently observed localized

to this region in interphase and mitotic cells, and ultrafine bridges can sometimes be observed

running between these foci during anaphase, suggesting that cells can enter mitosis with

unresolved intermediates, similar to what is observed with telomeric DNA in ALT cells (Chan et

al, 2009). Although treatment of cells with mitomycin C or aphidicolin increases the frequency

of cells with more paired FANCD2 foci on mitotic chromosomes, these foci are also observed in

untreated GM637 ALT cells (Chan et al, 2009). I have also frequently detected paired FANCD2

foci in undamaged GM847 and VA13 ALT mitotic cells that do not appear to colocalize with

telomeric DNA. The FRA16D locus is frequently found in micronuclei, suggesting that it is

unstable (Chan et al, 2009). Similar to ALT telomeric foci and endogenous S phase foci,

FANCD2 focus formation on paired mitotic sister chromatids is independent of ATM and ATR

(Chan et al, 2009). Given the association of FANCD2 with this fragile site, and the multiple

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characteristics that ALT telomeres share with expressed fragile sites, it would be interesting to

examine whether the FRA16D locus also becomes amplified as an extra-chromosomal element

and accumulates within PML bodies, and if this occurs in a FANCD2-dependent manner.

The presence of non-telomeric repeat DNA within APBs may be important because, if telomeres

interact with this DNA and non-telomeric sequences become integrated into the telomere itself, it

could result in impaired telomere capping ability due to a decreased ability of telomere binding

proteins to bind to these sequences. To date, a detailed analysis of the sequence makeup of ALT

telomeres has not been possible. However, experiments examining the first several kilobases of

ALT telomeres have shown a high degree of instability, which is not observed in telomerase

positive or primary cells (Varley et al, 2002). This analysis was performed using telomere variant

repeat-PCR, a technique which relies on amplification of genomic telomeric DNA in the

presence of radioactively labeled telomeric repeat sequences, and subsequent running out of the

products on denaturing PAGE gels followed by autoradiographic detection. Although the

hexameric TTAGGG repeat is the dominant telomere sequence, variations of this repeat

including but not limited to TGAGGG, TCAGGG, TTGGGG, CTAGGG, as well as pentameric

repeats, are present at elevated levels within the first ∼1.9kb of human telomeres (Allshire et al,

1989). Telomeres frequently contain a nonamplifying repeat type, which may be caused by the

presence of a variant not tested in the experiment, or a non-telomeric repeat (Varley et al, 2002).

Initial FISH experiments analyzing metaphases spreads for the presence of DNA from the MS32

satellite or FRA16D locus at telomeres could also be carried out in FANCD2-depleted of control

ALT cells.

ALT appears to be activated solely following a period of cellular crisis, when telomeres are short

and therefore contain a higher proportion of variant sequences. Additionally, short telomeres

may be more prone to rapid deletion and recombination events, which may result in an

overrepresentation of these variant sequences both within telomeres and the ECTR DNA.

Telomerase extension of short telomeres, which would normally act to add perfect TTAGGG

repeats to short telomeres after crisis, and thereby keep variant sequences constrained to the base

of the telomere, would not occur in ALT cells. Instead, ALT cells emerging from crisis have to

rebuild telomeres based on remaining short telomeres. If variant sequences are overrepresented

within rebuilt ALT telomeres, capping may be affected, as in vitro experiments suggest that

variants form different types of G-quadruplex structures and bind telomeric proteins with

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differing affinities (Lim et al, 2009; Mendez-Bermudez et al, 2009). In vivo evidence supports a

difference in telomere stability related to the presence of variant sequences (Mendez-Bermudez

et al, 2009).

To examine the level of variants within ALT telomeric DNA a FISH approach could be taken

using probes specific for the different variant telomeric sequences. Examination of interphase

cells for colocalization between variant sequences and APBs, would be informative for

determining whether or not these variants are present in ECTR DNA. The CTAGGG repeat

could be initially examined, as higher levels of this repeat have been associated with increased

mutation rates (Mendez-Bermudez et al, 2009). Quantitative-FISH analysis of signal intensity of

variant repeats in ALT metaphase telomeres could also be performed to determine if variants are

present at higher levels at ALT telomeres, relative to primary and telomerase telomeres.

Examination of ECTR DNA in metaphase spreads could also be used to confirm interphase cell

findings. FANCD2-depleted cells with elevated levels of telomeric recombination and ECTR

DNA would be a useful tool in these experiments, as rare phenomena may be easier to observe.

A more detailed picture of the structure of ALT telomeric DNA could be obtained using a

molecular combing approach with probes against TTAGGG and variant sequences.

In addition to causing potential differences in telomere capping, the presence of high levels of

variant telomeric sequences would mean that rather then recombination events occurring

between perfectly homologous TTAGGG sequences, ALT telomeric recombination could more

often be a form of illegitimate recombination involving homeologous sequences. In yeast cells,

mutations in the MSH2, MLH1, PMS1, or MSH3 and MSH6 mismatch repair (MMR) genes

promotes telomerase independent recombinational survival, possibly related to a loss of

suppression of illegitimate recombination between subtelomeric and telomeric sequences (Rizki

and Lundblad, 2001). A survey of human MMR deficient human cancers from individuals with

hereditary nonpolyposis colon cancer, and sporadic cancers both with and without microsatellite

instability failed to show a correlation between ALT activation and MMR deficiency, however

the types of cancers surveyed are not prone to activating the ALT pathway (Ibanez de Caceres et

al, 2004). A role for FANCD2 in helping to suppress illegitimate recombination events is

supported by examination of meiotic spreads from FA core complex and FANCD2 deficient

mice, which show frequent chromosomal mispairing, possibly occurring between imperfect

regions of homology (Wong et al, 2003; Houghtaling et al, 2003). Additionally, although BLM

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has activities that usually act to suppress recombination, in the context of homeologous

sequences it appears to stimulate recombination reactions (Kikuchi et al, 2009). Future

experiments to more directly test the role of FANCD2 in the regulation of illegitimate

recombination could also be carried out.

Telomere sequence differences in ALT versus telomerase positive or primary cells is one

possible explanation for difference in telomere stability, however additional factors may also

contribute. Aspects of ALT, such as rapid changes in telomere length, formation of extra-

chromosomal telomeric circular DNAs, and telomeres that have an altered staining pattern

during metaphase can also be induced in a non-ALT setting by modulating expression of

telomere capping components (Wang et al., 2004; Sfeir et al, 2009). This has led to the proposal

that ALT may be due to insufficient levels of shelterin telomere binding proteins, a hypothesis

supported by the fact that ALT cells have lower amounts of TRF2 relative to total telomeric

DNA content then non-ALT cells (Cesare et al, 2009).

In addition to potential insufficiencies in the shelterin complex, the relative expression of the

Ctc1/Sten1/Ten1 (CST) complex should also be examined within ALT cells by western blot

analysis. The CST complex is an RPA-like single-strand DNA binding complex, that in human

cells colocalizes with both telomeric and non-telomeric loci in interphase cells and does not

show binding specificity for telomeric sequence in vitro (Miyake et al, 2009). Although

colocalization of Sten1 with telomeric foci is not dependent on replication, Sten1 and Ten1 were

also characterized as proteins that that stimulate DNA polymerase-α-primase activity, and are

also referred to as AAF44 and AAF132 (Miyake et al, 2009, Casteel et al, 2009). Casteel and

colleagues showed general colocalization between myc tagged AAF44 and AAF132 with PCNA

during S-phase, and reduced replication following siRNA knockdown, suggesting a role for these

proteins in replication. Deletion of Sten1 or Ten1 from Arabidopsis thaliana results in severe

morphological abnormalities, sterility and telomere abnormalities in the first generation

knockout plants (Surovtseva et al, 2009; Song et al, 2008). Telomere abnormalities are not due to

changes in telomerase activity, and include increases in telomere length heterogeneity, increases

in single-strand G-rich DNA primarily at the overhang but also to a lesser extent at internal

regions of the telomere, accumulation of extra-chromosomal circular telomeric DNA, and

increased end fusions involving subtelomeric sequences (Surovtseva et al, 2009; Song et al,

2008). SiRNA knockdown of human CTC1 results in increased telomere free ends, chromatin

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bridging, formation of γH2AX foci in interphase cells, and increased single-strand G-rich DNA

both at the overhang and at internal sites (Surovtseva et al, 2009; Miyake et al, 2009). These

studies have led to the hypothesis that the CST complex may have a role in promoting replication

of difficult to replicate sequences, including telomeres. Insufficient amounts of these proteins at

ALT cells could explain both the telomere abnormalities, as well as potential abnormalities at

non-telomeric loci.

In this study I did not find evidence of telomere abnormalities in FANCD2 depleted telomerase

positive cell lines, however the uniform telomere length distribution and absence of critically

short telomeres, which appear to more frequently initiate recombinogenic events, could have

prevented detection of FANCD2-dependent phenotypes. A recent FANCC mouse study found

that in a TERT-/- background with increased levels of short telomeres undetectable by FISH,

FANCC deficient bone marrow cells have a higher frequency of T-SCEs then FANCC

expressing cells (Rhee et al, 2010). Increased frequencies of T-SCEs were not observed when

telomeres were longer, and the frequency of genomic SCEs was not increased in FANCC

deficient cells. As telomeres begin to shorten they may bind insufficient amounts of shelterin

components to fully cap ends, making them more prone to recombination events. Additionally,

as telomeres shorten they are more likely to have higher frequencies of variant sequences, which

may further alter shelterin binding as well as the capacity of telomeres to form t-loops. In this

short telomere setting telomeric recombination may often involve homeologous sequences, and it

is in this situation of potential illegitimate recombination events that the FA pathway may play a

critical role in regulating recombination.

To investigate whether FANCD2 has a telomere length dependent role in suppressing telomeric

recombination in human cells, I would propose examining the effect of FANCD2-depletion on

telomeric recombination in primary BJ cells from different passages. The status of telomeres in

BJ cells has been well characterized, and I currently have E6/E7 expressing BJ cells from

population doublings 30-95 (Zou et al, 2004; Zou et al, 2009). As these cells express E6 and E7

oncogenes, they can continue to replicate past mortality stage 1, into a period of telomere

induced cellular crisis with a high frequency of critically short telomeres. The expression of E6

and E7 oncogenes will allow cells that may normally not enter mitosis, to continue cycling and

their telomeres to be assayed. Early, mid and late passage FANCD2 and random siRNA treated

cells could be examined for the presence of T-SCEs, telomere free ends, and changes in the ratio

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of telomere lengths on the p and q arms of chromosomes. Additionally, I have previously

generated FANCD2 targeting shRNA vectors that have been successfully used to generate stable

clones with reduced FANCD2 expression levels. Although extreme levels of FANCD2 silencing

in ALT cells severely reduces cell viability, it is possible that more modest decreases in

FANCD2 levels during the period of cellular crisis may act to encourage activation of the ALT

pathway. To test this hypothesis, FANCD2 shRNA BJ E6/E7 expressing clones could be

generated, and then followed through crisis and the telomere status of emerging immortalized

cells could be examined.

This work has explored the role of the FA pathway in telomere maintenance. Although I did not

find evidence to support a causal role of telomere maintenance defects in the pathogenesis of FA,

I have obtained results that have helped to advance our understanding of both the ALT process

and the role of FANCD2 within recombination. The additional experiments proposed in this

section will act to expand our understanding of both the function of the ALT pathway, and the

role of FANCD2 in DNA repair.

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