Molly King - Thesis Final

7/31/2019 Molly King - Thesis Final

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An investigation into the interaction ofX. laevis telomeric proteins TRF1 and PinX1

A Thesis

Presented to

The Division of Mathematics and Natural Sciences

Reed College

In Partial Fulfillment

of the Requirements for the Degree

Bachelor of Arts

Molly McCarthy King

May 2009


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Approved for the Division

(Biology)

Janis Shampay


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Acknowledgments

I would like to thank my advisor Janis Shampay for believing that one day, I

would, in fact, actually make it into lab, and for your willingness to answer all of my

stupid questions when I did. Janis, I am so grateful for your infinite patience,

understanding, and encouragement that reinvigorated my love of science.

Mom and Dad, you are the reasons I made it to Reed, and the reasons I made it

out. Thank you for the incredible sacrifices you made to get and keep me here. Thank

you for making me get back on the plane, especially that first time. Thank you for

answering my calls at 2 am, and understanding when I failed to call for weeks. Words

can never thank you enough for all you have done to help me be me.

I would like to thank all of my friends at Reed for getting me through these long

days, weeks, years. Knowing that you were in this with me made each day doable, even

when I sometimes didnt see you for months at a time. Annie Miano, Taiga Christie (my

other two Musketeers), Sarah Kemp, Pooja Bhaskar, Anna McGee, and Cori Savaiano: I

am so blessed to have gotten to know you. You are incredibly special people, and

incredibly special to me. I couldn't have made it through Reed without you girls, no way.

I feel so honored to call you my best friends your unconditional love and support mean

so much to me. Adarsh Pyarelal, thank you for putting up with my crazy, for giving me

the support and understanding to endure this year, for sharing your love and contagious

enthusiasm for life, and for making this past year happier than I thought possible. Thank

you to my role models and friends who went before me, Christine Lewis, Allison Edgar,

and Courtney von Bergen, who showed me the path to involvement, passion and balance,

showed me it is possible, and helped keep me sane through the darkest of times. To my

wonderful friends, Matt Jemielita, Rachel Cooper, Maeve Hooper, Andrew Betson, Nick

Bradish, Kevin Lynagh, Ryan Gersovitz, Celia Hassan, Michael Reisor, Jeryl Hewey,

Lee Lipton, Kailyn McCord, Sean Lerner, Dana Bublitz, Chris Williams, AmritaRajasingham, Catherine Hinchliff, Advait Jukar, Javed Parkes: thank you all for your

camraderie, love, and infinite understanding of my very human failings throughout the

years I couldnt have done it without you, my Reed family, and I certainly would not

have had this much fun.


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To my mentors, thank you for taking me under your respective wings. Noelle

Faricy, Kristin Holmberg, Kyle Webster and the SAO, Gabe Merrell and Amy Frey,

thanks for all of your insight, patience, guidance and encouragement, for listening non-

judgmentally to my rants, and for the always-warm welcomes to a much-needed refuge

from academia and everything else. Thank you Kjersten Whittington, for giving me

direction and hope that there is something for me at the intersection of science and society,

and for your belief in me as an individual and as an academic. Thank you Bob Kaplan, for

always being there and for encouraging and supporting me, even in those times when I

wasnt able to make academics priority one. To the mentors who got me to Reed, Donna

Gordon and Mike Erikson, I can never thank you enough for all you did to help me grow

in high school. Thank you all, I would have been lost without your guidance.

To my lab-mates Dave Constant and Jen Jin: thank you for all of your help when I

was utterly clueless, for your late-night solidarity, and for your friendship in and outside

of lab. A huge thanks to Mark Amoruso for all your help and encouragement in lab.

A bittersweet thanks to Senate, which was such a big part of my identity at Reed,

for making me truly appreciate the moments when I got to be a student and for the

invaluable lessons I learned outside of the classroom. I am grateful for having the

opportunity to work with a group of people as dedicated to and passionate about Reed as

my senators, and I never could have made it through a year of thesis with student body

presidenting without you. Ajax/Overmind/Bradish, I couldnt have done it without you

thanks for being such a super president of vice and partner in crime. And thank you to the

administrators Mike Brody, Lily Copenagle, Barre Stoll, Colin Diver, Kathy Rose,

Michael Leidecker, Towny Angell, and again, Kristin Holmberg who were willing to

listen and collaborate, and who work together year after year to keep this institution the

place I have grown to love.

Also, a silly but genuine thanks to all the inanimate objects and ideals in my life

that helped me through coffee, 3 am conversations about the Honor Principle, xkcd and

PhD comics, the Senate Bylaws, and a belief that someday, all this will be worth it. And

the realization that, because it is about the process and not the product, that day is already

here.

This research was supported in part by a grant from the Reed College Biology

Undergraduate Research Project Program.


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Table of Contents

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

The problem with telomeres ........................................................................................1

Telomeres: the chromosome protection complex......................................................1

Telomere structure...................................................................................................1

Duplicating chromosomes: the end replication problem...........................................2

The DNA damage response......................................................................................4

DNA repair pathways ..............................................................................................4

T-loops: distinguishing chromosome breaks from telomere ends..............................5

Telomerase maintains telomere length .....................................................................7

The shelterin complex and associated factors...............................................................8

TRF1 (Telomeric Repeat binding Factor 1)............................................................10TRF2 (Telomeric Repeat binding Factor 2)............................................................12

POT1 (Protection of Telomeres 1) .........................................................................13

TPP1 (TINT1 / PTOP / PIP1).................................................................................14

TIN2 (TRF1-Interacting Nuclear protein 2)............................................................14

RAP1 (Repressor/Activator Protein 1) ...................................................................15

PinX1 is a TRF1-interacting telomerase inhibitor ...................................................... 15

The roles of telomeres, TRF1 and PinX1 in oncogenesis ...........................................18

The interaction of PinX1 and Trf1 .............................................................................18

Xenopus as a model organism....................................................................................19

Experimental goals and design...................................................................................20

Materials & Methods...................................................................................................23

Sequence alignment analysis .....................................................................................23

PCR construction of Myc-tagged xlTRF1dd ..............................................................23

Primer design.........................................................................................................23

Amplification of desired construct .........................................................................24

Subcloning of xlTRF1dd PCR product into intermediate vector.................................25

PCR product purification .......................................................................................25Subcloning and transformation ofE. coli ...............................................................25

Small plasmid DNA isolations............................................................................... 26

Analysis of isolated plasmid DNA.........................................................................26

Construction of pTNT in vitro translation clone.....................................................27

Ligation into pTNT vector .................................................................................27


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Transformation ofE. coli with pTNT-xlTRF1dd vector......................................... 28

pTNT-xlTRF1dd plasmid purification................................................................... 28

Sequence analysis of plasmid DNA........................................................................... 29

In vitro expression of proteins................................................................................... 29

Immunoprecipitation................................................................................................. 30

Coimmunoprecipitation............................................................................................. 30

Visualization of proteins ........................................................................................... 31

SDS-PAGE separation of protein products ............................................................ 31

Visualization of total protein ................................................................................. 31

Electroblotting....................................................................................................... 32

Detection of biotin labeled amino acids ................................................................. 32

Detection of His-tagged protein on western blot .................................................... 32

Results & Discussion................................................................................................... 35Overview.................................................................................................................. 35

In silico protein sequence analyses............................................................................ 35

TRF1 sequence analysis ........................................................................................ 35

PinX1 sequence analysis ....................................................................................... 37

Interaction with xTERT......................................................................................... 39

Design of experimental constructs............................................................................. 39

PinX1 construct design.......................................................................................... 39

Primers amplify dimerization domain from full-length xlTRF1 clone.................... 39

Intermediate subcloning of xlTRF1dd-Myc construct................................................ 40

Transformed bacteria contain intermediate vector with insert ................................ 41

Purified xlTRF1dd-Myc construct inserted into in vitro translation vector................. 42

Transformed bacteria contain expression vector with desired insert ....................... 43

Sequence analyses confirm no mutations in xlTRF1dd construct............................... 45

pTNT-xlTRF1dd clone j used for in vitro translation................................................. 45

Visualization of protein products............................................................................... 46

Immunoprecipitation................................................................................................. 48

Coimmunoprecipitation experimental design............................................................. 49Coimmunoprecipitation attempt ............................................................................ 52

Conclusions and recommendations for further study ................................................. 52

Appendix A: Sequence of Myc-tagged xlTRF1dd protein construct ........................ 55

Bibliography................................................................................................................ 57


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List of Tables

Table 2.1. PCR primers used to create Myc-tagged xlTRF1dd construct........................23

Table 2.2. Cycling program for amplification of xlTRF1dd. .......................................... 24


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List of Figures

Figure 1.1. Chromosome replication results in the end replication problem, gradually

shortening successive daughter strands without the remediative action of telomerase.

................................................................................................................................3

Figure 1.2. The t-loop at the ends of chromosomes is theorized to form via the invasion of

the G-rich strand into the internal complementary sequence of the C-rich strand......5

Figure 1.3. Domain diagrams of human shelterin and PinX1 proteins. .............................9

Figure 1.4. The protein components of the shelterin complex bound to the telomeric

overhang................................................................................................................10

Figure 3.1. TRF1 amino acid sequence alignment among species.. ................................36

Figure 3.2. PinX1 amino acid sequence alignment among species. ................................37

Figure 3.3 Cross-species alignment of domain of PinX1 that interacts with TRF1dd......38

Figure 3.4. Myc-tagged xlTRF1dd construct resulting from amplification by designed

primers. . ...............................................................................................................40

Figure 3.5. Insertion of xlTRF1dd-Myc construct in intermediate cloning vector...........41

Figure 3.6. Analytical gel to confirm restriction digests and estimate concentration of

purified TOPO-xlTRF1dd DNA.............................................................................42

Figure 3.7. Desired pTNT-xlTRF1dd-Myc in vitro translation construct.......................43

Figure 3.8. Restriction enzyme analysis of clones obtained from plasmid purification of

pTNT-xlTRF1dd samples. .....................................................................................44

Figure 3.9. Sequenced 5 and 3 terminal fragments of xlTRF1dd-Myc construct. .........45

Figure 3.10. Visualizations of protein products from in vitro transcription/translation

reactions of xlTRF1dd-Myc and xlTRF1full-Flag..................................................47

Figure 3.11. Biotin labeled amino acids obtained from immunoprecipitation. ................49

Figure 3.12. Schema of experimental design for IP and co-IP reactions. ........................ 51


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Abstract

The chromosome replication process causes the ends of chromosomes to slowly

degrade without the remediative action of the enzyme telomerase. Telomeres are

repetitive sequences at the ends of chromosomes that protect them from inappropriate

DNA repair and cellular responses to DNA damage. Telomerase and other telomere-

associated proteins are essential for end protection, chromosome maintenance and

telomere regeneration. Two telomere protection complex proteins, TRF1 and PinX1,

have been found to interact and to negatively regulate telomere length in humans.

Human PinX1 is a direct inhibitor of telomerase enzymatic activity, while TRF1

indirectly regulates telomerase by preventing access to the telomeric overhang. Xenopus

laevis has previously been shown to have constitutive telomerase activity in somatic

cells, making it an excellent model organism for research on telomerase and telomerelength regulation. This study sought to investigate the interaction of the telomeric

proteins TRF1 and PinX1 inXenopus laevis. An in vitro expression construct for a Myc-

epitope tagged TRF1 dimerization domain was created to aid in the study of this

interaction. This construct can be used in future studies for more specific identification

of interacting domains, should PinX1 and full-length TRF1 be found to interact inX.

laevis. The interaction ofX. laevis PinX1 and full-length TRF1 was investigated using

previously cloned constructs. Immunoprecipitation and coimmunoprecipitation studies

were performed to investigate in vitro interaction. While no conclusive evidence was

found either for or against interaction, progress was made towards a rigorous

experimental design to test this interaction.


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Dedication

For my parents, who were right all along.


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Introduction

The problem with telomeres

Telomeres: the chromosome protection complex

Chromosomes are long molecules of DNA and histone proteins that contain an

organisms genetic material. The ends of eukaryotic chromosomes are protected by

telomeres, nucleoprotein structures that regulate telomere replication and safeguard

against degradation. Since eukaryotic chromosomes are linear, their ends are especially

vulnerable to degradation by enzymes (Palm and de Lange, 2008). Their linearity poses

particular challenges in chromosome replication processes, an issue known as the end

replication problem. Normal telomere function is essential for cell viability. Without

sufficient telomere protection, shortening of telomeres can result in chromosome loss and

end-to-end fusions (Evans and Lundblad, 2000).

Most eukaryotes have telomeres composed of double-stranded short tandem DNA

repeats. In vertebrates, this sequence is TTAGGG (Meyne et al., 1989; Blasco, 2005).

Telomere-associated proteins consist of the shelterin complex and the accessory factors it

recruits, to be described in more detail below.

Telomeres and their associated proteins serve four capping functions for

chromosomes: regulation of telomere length; prevention of the DNA damage response to

telomere presence; prevention of the fusion of chromosome ends; and prevention ofhomologous recombination between telomeric DNA (Chan and Blackburn, 2004). These

roles are vital for chromosome stability and cell survival and proliferation.

Telomere structure

The telomere length necessary to adequately protect chromosomes varies among

organisms. Human telomeres are usually 10-15 kb (kilobases), but also contain

additional TTAGGG repeats in subtelomeric regions (Palm and de Lange, 2008).

Telomeres in the model organism for this study,Xenopus laevis, range from 10-50 kb in

length and can vary among tissues in the same individual (Bassham et al., 1998).

Due to their composition of TTAGGG repeats, telomeres have a 3 strand rich in

guanosine and lacking in cytosine. The two complementary strands of the telomere are

thus referred to as the G-strand and the C-strand (Palm and de Lange, 2008). The G-

strand of eukaryotes typically ends in a 100-200 nucleotide single-stranded overhang


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(Blasco, 2005; De Boeck et al., 2009), a protrusion that is generally longer but more

variable (50-500 nt) in mammals (Palm and de Lange, 2008).

Duplicating chromosomes: the end replication problem

The linear nature of eukaryotic chromosomes presents a grave problem for themaintenance of these genetic-programming structures. The chromosome replication

machinery necessarily leaves a single-stranded overhang at each end after replicating the

linear DNA (Figure 1.1). DNA is replicated by a process described by Watson and

Cricks semiconservative model, meaning that during each replication cycle, the two

copies of DNA will each contain one strand from the original chromosome and one

daughter strand. Replication occurs continuously for one strand the leading strand

and discontinuously for the other the lagging strand (Lander, 2005). The progressing

replication fork is supported by telomere proteins, permitting efficient telomere synthesis

(Verdun and Kalseder, 2007).

During DNA replication processes, the enzyme primase synthesizes a single-

stranded RNA primer at the 5 end of replication. DNA polymerases then extend the

strands initiated by RNA primers in the 5' to 3' direction. But DNA polymerase can only

synthesize the daughter strand in the 5 to 3 direction. When the leading RNA primers at

the 5 end of the newly replicated chromosome are removed, this leaves a single-stranded

overhang that cannot be filled by DNA polymerase. Without the action of telomerase to

lengthen the telomeric regions, the process of DNA replication results in progressively

shorter and shorter daughter molecules (Lander, 2005).At the end of replication, the other end of the chromosome, resulting from

leading-strand synthesis, will theoretically be blunt. The formation of a functional

telomere structure and the binding of the necessary proteins require a single-stranded

overhang (Verdun and Kalseder, 2007). The sequence loss observedduring cell

replication is greater than that predicted by the end replication problem alone (Harley et

al., 1990). Detection of such long overhangs at both ends of a chromosome suggests that

there are regulated processing methods for single-stranded overhang generation, in

addition to the overhang resulting from the end replication problem. It is proposed that

the G-rich daughter strand overhang may be generated at the blunt end by nucleolytic

digestion in the 5' to 3' direction, though no nuclease candidates have yet been identified

(Verdun and Kalseder, 2007).


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Figure 1.1. Chromosome replication results in the end replication problem, gradually

shortening successive daughter strands without the remediative action of telomerase.


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The DNA damage response

The average lengths of telomeres are far in excess of those needed merely to

protect chromosome function. In humans, a minimum of 78 bp (13 repeats) of TTAGGG

sequence is required to prevent fusions of telomeres. A fully functional telomere can be

developed from as few as 400 bp of TTAGGG repeats. Telomere length must be reduced

to less than 1000 bp to cause senescence in human tumor cell lines (Palm and de Lange,

2008). Because of this length requirement for functional protection, embryonic-tissue

derived human cells can only divide about 50 times before shortened telomeres cause the

cell to permanently arrest division or die. This division boundary, known as the Hayflick

limit, is determined by the rate of shortening and the initial telomere length (Verdun and

Kalseder, 2007).

Telomeres that become too short are detected by the cells damage response

pathway as a double-strand break. The pathways of cell senescence and apoptosis areassociated with shortened telomeres (Lechel et al., 2005). As discussed above, during

each cell replication, telomeres are shortened as a result of the end replication problem.

In the absence of corrective telomerase action, telomere erosion limits the proliferative

potential of cells through either apoptosis or senescence (Palm and de Lange, 2008).

When telomeres are dysfunctional, the DNA damage response is initiated through

one of two pathways dependent on one of the protein kinases, ATM or ATR. Double

strand breaks activate the ATM pathway, while the ATR pathway responds to the

formation of single stranded DNA. Activation of DNA damage response pathways

results in an arrest of cell proliferation (Palm and de Lange, 2008). This leaves telomeres

susceptible to inappropriate DNA repair or cells vulnerable to senescence.

DNA repair pathways

Double strand DNA breaks in mammals are repaired by either nonhomologous

end joining (NHEJ) or homology directed repair / homologous recombination (HR)

(Palm and de Lange, 2008). When telomeres are left unprotected, or the telomere

protection complex malfunctions, chromosomes are threatened by these repair

mechanisms.

Homologous repair occurs when the homologous region of a sister chromosome

acts as a template to guide repair of the broken strand. Repair begins with the creation of

a single-stranded overhang which then invades a homologous sequence to form a hybrid

heteroduplex (De Boeck et al., 2009). The protein complex shelterin that localizes to

telomeres helps protect against the three kinds of HR: t-loop HR, HR between sister

telomeres, and HR between a telomere and interstitial telomeric DNA (de Lange, 2005).


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In NHEJ, two double strand breaks are joined directly without regard for

sequence homology (De Boeck et al., 2009). ATM/ATR signaling is required for NHEJ

to fuse chromosome ends (Palm and de Lange, 2008). Double-strand breaks are

recognized by the binding of the Ku70 and Ku80 proteins and the MRN complex. The

association of these factors recruits the catalytic subunit of DNA protein kinase (DNA-

PK), which then initiates a pathway leading to repair of the break by DNA ligase (De

Boeck et al., 2009). The double-strand break repair protein DNA-PK has a dual function

in capping telomere ends. Cells lacking the catalytic subunit of the DNA-PK protein

exhibited substantial numbers of inappropriate end-to-end fusions, implicating this

protein in telomeric end-capping, an additional function to its role in repairing incidental

DNA damage (Bailey et al., 1999). Inhibition of ATM kinase activity led to telomere

dysfunction, suggesting that the formation of the chromosome end protection complex

actually depends upon the post-replication DNA damage response at telomeres (Verdun

et al., 2005).

The structure of telomeres leaves them vulnerable to these reactions to damage,

resulting in inappropriate DNA repair or cell senescence. This necessitates some means

by which to distinguish telomeric DNA from damaged DNA.

T-loops: distinguishing chromosome breaks from telomere ends

One possible mechanism by which the DNA damage response machinery

distinguishes telomere ends from chromosome breaks in mammals is via telomere

formation into the telomeric loop (t-loop), structure. The t-loop structure forms both invivo and in vitro by invasion of the 3 overhang into the double-stranded segment of

telomeric repeats. The invading 3 G-strand overhang supplants the G-strand in that area

to form the displacement loop, or D loop (Figure 1.2).

Figure 1.2. The t-loop at the ends of chromosomes is theorized to form via the invasion of

the G-rich strand into the internal complementary sequence of the C-rich strand.

This structure may be responsible for telomere protection from both telomerase extension

and enzymatic degradation.


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Although this structure has been observed in vivo, there is no conclusive experimental

evidence that it is necessary for protecting telomeres from the DNA damage response.

T-loop structure formation is mediated by the shelterin protein TRF2 (Griffith et

al., 1999). TRF2 is found at the loop junction, preferentially binding to the area between

the single-stranded overhang and the duplex loop (Stansel et al., 2001). However, the

role of TRF2 in forming t-loops is unlikely due to its DNA binding function alone;

crystal studies of TRF2-DNA complexes indicate that the DNA binding domain of TRF2

binds double-stranded DNA without any major distortions of the DNA (Court et al.,

2004). TRF2 mediates DNA loop formation at any point in the telomeric repeat sequence

by forming a tetramer at the branching point of the loop. Though in vitro TRF2-dependent

loop formation does not require the 3 overhang that characterizes telomeres, the

formation of an end-loop (as opposed to a loop at any point in the repeat tract) requires

the specific sequence of the overhang (Yoshimura et al., 2004). The tumor suppressor

protein p53 also plays a role in in vitro t-loop formation, increasing the efficiency of

TRF2 t-loop formation by two fold when it is present at the t-loop junction (Stansel et al.,

2002).

Random breaks in the chromosome will be less capable of forming protective t-

loop structures, as they lack the highly repetitive sequences that promote the invasion of

one strand by another. Furthermore, short telomeres will be less able to form t-loops, and

thus cells with highly degraded chromosomes will not be inappropriately protected from

the natural course of the DNA damage response pathways (Griffith et al., 1999).

However, t-loops can form from as little as 500 bp and range up to 18 kb in length in

mammals (Wei and Price, 2003). The size of the loop varies by species and is correlated

with overall telomere length, with t-loops being generally a few kb shorter than the total

telomeric DNA (Wei and Price, 2003).

Griffith et al. (1999) suggest that the t-loop may be responsible for protection

from telomerase extension and enzymatic degradation in vivo. This speculation is

corroborated by findings that telomere chromatin extracted from human, mouse and

chicken nuclei forms t-loops (Griffith et al., 1999; Nikitina and Woodcock, 2004). Thus,

the t-loop model presents one possible mechanism for distinguishing telomeres from

genuine double strand breaks. The characterization of another equally effective

protective structure, comprised of two proteins that bind the ends of chromosomes in

Oxytricha nova, reinforces the idea that t-loops are only one (albeit rather attractive)

model for vertebrate telomere protection (Verdun and Kalseder, 2007).


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Telomerase maintains telomere length

Telomerase, initially known as telomeric terminal transferase, is a reverse

transcriptase that extends the length of the 3 G-strand following chromosomal

replication. The activity of telomerase is one example of a reversal of the central dogma

of biology, which states that the directionality of synthesis proceeds from DNA to RNA

to protein. Telomerase uses an RNA template to synthesize replacement DNA repeats at

the terminal ends of telomeres (Chan and Blackburn, 2004).

Telomerase has a protein component (TERT) and an RNA sequence component

(TR) that it uses to prime DNA synthesis. In humans and yeast, telomerase forms a

dimeric complex, but it is undetermined whether this dimerization is a conserved or

essential characteristic of the enzyme. Although the protein component of telomerase is

responsible for catalyzing the polymerization reaction, it has been shown that TR RNA

template mutations can have an effect on enzymatic function. A trinucleotidesubstitution in the TR template domain ofS. cerevisiae destroys the enzymatic function

of telomerase, indicating that the template region requires base-specific interactionsfor

functional enzyme activity (Chan and Blackburn, 2004).

Telomerase lengthens the 3 single-stranded overhang of telomeric DNA. The

distal nucleotides of this strand base pair with the complementary TR component of

telomerase. The strand is extended by polymerization of additional G, T and A

nucleotides using the TR RNA as a template. Telomerase releases this newly synthesized

segment, which is then available either for further elongation by telomerase or for

complementary lagging-strand synthesis of the C-strand using the newly synthesized G-

strand region as a template for primase-polymerase (as described above in the

chromosome replication process) (Chan and Blackburn, 2004).

In this way, the function of telomerase opposes the losses to telomere length due to

the end replication problem and other DNA damage. Some minimal number of telomeric

repeats is necessary for the foundation of the telomerase-shelterin complex that protects

the telomere. The length of telomeric repeat tract seems to be one determinant of whether

or not telomerase can access the chromosome end; shortened telomeres are uncapped, or

accessible to telomerase, while elongated telomeres are capped, or telomerase-inaccessible. Initially, this might imply the conclusion that telomerase has no function at

telomeres until they reach a critically short length (Chan and Blackburn, 2004).

However, this does not appear to be the whole story. Telomerase appears to play

an important role in preventing inappropriate repair by the DNA damage response. Rather

than merely the presence of long telomeres and t-loops, it is in part the attendance of


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telomerase that protects telomeres from fusions. Instead of promoting end-joining, the

DNA damage response factors attracted to telomeres actually promote telomerase action

(Chan and Blackburn, 2004). In one possible model, chromosomes are regularly cycling

through end-capped and -uncapped forms, with the DNA damage response as an important

feedback mechanism for promoting telomerase activity (Chan and Blackburn, 2004).

Telomerase activity is also connected with the cell growth and division cycle, with

the protein only disconnecting from the telomeres during mitosis. This explains the role of

telomerase in maintaining adequate telomere length to ensure cell proliferation (Chan and

Blackburn, 2004). InXenopus egg extracts,the activity of telomerase was higher when

associated with interphase chromatin than with mitotic chromatin. This finding suggests

that telomere chromatin is actively regulated by processes dependent on the timing of the

cell cycle, including recruitment of telomerase to the replicating chromatin during

interphase (Nishiyama et al., 2006). Telomerase is expressed ubiquitously inX. laevis

somatic cells, with differential activity in different tissues not solely due to differential

amounts of diffusible telomerase inhibitor (Bousman et al., 2003).

A lack of telomerase activity in cells results in telomere shortening, reduced

proliferation and, eventually, cell senescence. Even within a single cell, telomere lengths

are highly heterogeneous, but the overall population of telomeres in a group of dividing

cells is kept within limits specific to that cell type (Chan and Blackburn, 2004). The

limited amount of telomerase in human cells maintains telomeres at a certain equilibrium,

and a low concentration of telomerase is critical for preferential lengthening of short

telomeres. One in vivo study showed that overexpression of the limiting hTERT and hTR

telomerase components resulted in a greater association of telomerase with telomeres and

their consequent elongation at a constant rate independent of telomere length (Cristofari

and Lingner, 2006). This equilibrium is maintained via the regulation of telomerase

access to telomeres (Evans and Lundblad, 2000). A complex called shelterin and other

associated proteins bind to telomeres, playing a role in telomere structure and in the

regulation of telomerase.

The shelterin complex and associated factorsAlso referred to as the telosome, shelterin is a complex of six proteins that bind to

telomeres, protect them from recognition by double-strand break machinery, and regulate

their length (De Boeck et al., 2009). These proteins are TRF1, TRF2, TIN2, TPP1, POT1,

and RAP1 (Figure 1.3). The shelterin complex also recruits other protein factors, among

which is TRF1-interacting PinX1.


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Figure 1.3. Domain diagrams of human shelterin and PinX1 proteins.

Matching colors correspond to either similar domain structures or protein-protein

interaction domains (de Lange, 2005; Palm and de Lange, 2008). Domain structure

acronyms for each protein are as follows, from the N terminus to the C terminus:

TRF1: Tankyrase-binding D/E-rich domain, also known as the acidic domain (white);

TRFH (TRF Homology) dimerization domain (blue); SANT/Myb DNA-

binding domain (green)

TRF2: GAR(Gly/Arg-rich) domain, also known as the basic domain (white); TRFH

(TRF Homology) self-dimerization domain (blue); RAP1 interaction domain

(brown); TIN2 interaction domain (orange); SANT/Myb DNA-binding domain

(green)

POT1: OB (oligonucleotide/oligosaccharide/oligopeptide binding) folds (shades of red);

TPP1-binding domain (yellow)

TIN2: TPP1 and TRF1 interaction domain (purple and orange); TRF1-binding domain

(blue)

TPP1: OB fold / potential telomerase interacting motif (red); POT1 interaction domain

(yellow); Ser-rich region (white); TIN2 interaction domain (purple)

RAP1: BRCT (BRCA1 C-terminal) protein interaction domain (white); Myb domain

(green); TRF2 interaction domain (brown)

PinX1: G-patch (glycine-rich RNA-interacting domain) (white); TERT nucleolar

localization domain (white); TID (telomerase inhibitory domain) (white)


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The proteins interact with each other and numerous other factors to regulate the

actions of telomerase, prevent DNA damage response signaling, and maintain telomere

structure. Shelterin specifically binds telomeric DNA due to the specificity of its proteins

TRF1, TRF2, and POT1 for the telomeric repeat sequence TTAGGG (Figure 1.4) (Palm

and de Lange, 2008). POT1 binds the single stranded region of the G-strand overhang

and in the D-loop within the t-loop structure. TRF1 and TRF2 bind the duplex region of

telomeres and recruit the other four components of the shelterin complex. Even in the

absence of telomeric DNA in nuclear cell extract isolations, shelterin forms a stable

complex (Palm and de Lange, 2008).

Figure 1.4. The protein components of the shelterin complex bound to the telomeric

overhang (used with permission, de Lange, 2005).

TRF1 (Telomeric Repeat binding Factor 1)

TRF1 is a 56 kDa telomeric protein that recruits shelterin-associated factors and

negatively regulates telomere length. The mammalian protein has an acidic domain at its

N-terminus, followed in sequence by a TRF homology (TRFH) dimerization domain near

its N-terminus and a C-terminal DNA-binding SANT-Myb-like domain (Chong et al.,

1995; Bianchi et al., 1997). The dimerization and Myb domains are joined by a highly

flexible linker of about 100 aa (Court et al., 2004). TRF1 binds double-stranded DNA in

vivo (Smogorzewska et al., 2000).

The C-terminal Myb-like domain, consisting of residues 371433 in human TRF1

(Hanaoka et al., 2005), is responsible for this binding of TRF1 to double-stranded DNA.The Myb domain recognizes two repeats of the GGGTTA sequence for selective binding

(Konig et al., 1998). Although TRF1 is able to bind DNA as a monomer, two Myb-like

domains can bind 6 bp apart (Konig et al., 1998), and TRF1 homodimers require the Myb

domains of both proteins for high-affinity binding (Bianchi et al., 1997). TRF1 binds

double-stranded telomeric DNA as a homodimer, formed by the interaction of the


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TRFH/dimerization domains of each protein. As a consequence of this interaction, the

TRF1 homodimer bends the site of its telomeric DNA to an angle of approximately 120

(Bianchi et al., 1997). The two Myb-like domains of dimerized TRF1 can independently

bind two recognition sites with great spatial flexibility (Nishikawa et al., 2001). This

function could contribute to the overall structure of the telomeric complex (Bianchi et al.,

1997). Though there may be some cooperative interaction between TRF2 and TRF1 in

end loop formation, the presence of TRF1 does not directly mediate the formation of t-

loops (Yoshimura et al., 2004).

TRF1 is a negative regulator of telomere length. One study in human cell lines

showed that preventing TRF1 binding prompts telomere lengthening, while TRF1

overexpression in telomerase-positive cells causes telomere shortening. A TRF1 clone

lacking DNA binding ability was created via deletion of the Myb domain. This

dominant-negative mutant heterodimerizes with coexpressed wild-type TRF1 and

prevents it from binding telomeric DNA, resulting in lengthening of telomeres (van

Steensel and de Lange, 1997). TRF1 regulates telomerase via physically preventing

access to the telomere, rather than by affecting telomerase expression levels

(Smogorzewska et al., 2000).

TRF1 is itself regulated by the ADP-ribose polymerase, tankyrase. Tankyrase

removes TRF1 from telomeres, allowing access to telomerase and thereby promoting

telomere elongation (Smith et al., 1998). Human tankyrase binds the human TRF1

(hTRF1) sequence RXXPDG contained within the D/E-rich acidic domain (Sbodio and

ChiDagger, 2002). Xenopus laevis andXenopus tropicalis both have homologs of TRF1

with substantially shorter D/E-rich acidic domains (Crumet et al., 2006). This truncated

acidic domain has implications for the regulation ofXenopus TRF1 by tankyrase, as it

does not contain the RXXPDG sequence.

The 49 kDaXenopus TRF1 protein has previously been cloned and characterized,

the most distantly related vertebrate genes from mammals that have been characterized to

date (Crumet et al., 2006; Nishiyama et al., 2006). The Myb domain is characterized by

76% identity to human TRF1 Myb domain (Smogorzewska and de Lange, 2004), and the

dimerization domain shares 49% identity and 68% similarity with the human equivalent

(Crumet et al., 2006).

Overexpression of dominant-negative TRF1 causes increased telomere elongation

in cultured human cells. Cell viability is not affected by the inhibition of human TRF1

with a dominant-negative allele (van Steensel and de Lange, 1997). But the surprising

finding that deletion of the first exon of mouse TRF1 results in rapid embryonic lethality

suggests an essential function for TRF1 that is independent of its role in telomere length


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regulation (Kalseder et al., 2003). Deletion of TRF1 results in activation of the ATR-

dependent DNA damage response because of its role in recruiting other shelterin factors.

Recruitment by TIN2-TRF1 is crucial for POT1 to repress the ATR response (Sfeir and

de Lange, unpublished data in Palm and de Lange, 2008). TRF1 deletion results in a loss

of TIN2 and TRF2 association with the telomere (Iwano et al., 2004). It could be this

loss of TRF2 that causes embryonic lethality as a result of TRF1 deletion.

Another possible lead on this telomere-length-independent function is the role

TRF1 plays in preventing activation of the ATM DNA damage response pathways.

TRF1/Pin2 (very similar proteins likely encoded by the same gene)

coimmunoprecipitates with ATM kinase. ATM kinase phosphorylates TRF1/Pin2 in

vitro and in vivo, inhibiting TRF1/Pin2 from carrying out its normal function of mitotic

induction (Kishi et al., 2001b). This interaction between ATM kinase and TRF1 is

notable because DNA damage response factors recruited to telomeres also promote

telomerase action (Chan and Blackburn, 2004). This may imply some involvement of

ATM damage response factors in regulating TRF1s function as a negative regulator of

telomerase.

TRF1 has additional functions in the cell cycle. InXenopus embryos, TRF1

associates with mitotic chromatin, an interaction promoted by TRF1 phosphorylation by

the mitotic kinase Plx1. Xenopus TRF1 dissassociates from chromatin during the

transition into interphase, a process coupled with reduced Cdc2 kinase activity and

increased telomerase activity. This is consistent with the finding presented earlier that

the activity ofXenopus telomerase is higher in associations with replicating interphase

chromatin than with mitotic chromatin, as the presence of TRF1 throughout mitosis

would exclude telomerase association (Nishiyama et al., 2006).

This finding stands in contrast to other studies that have shown that hTRF1

associates with telomeres throughout the cell cycle. Chromatin immunoprecipitation

experiments showed that hTRF1 shows a 50% decrease in telomere binding at the start S

phase, an increase in binding at the end of the S phase, and then an approximate 20%

decrease in binding in G2 (Verdun et al., 2005). One reason this may differ from the

TRF1-telomere associations inXenopus is becauseXenopus embryos alternate between S

and M phases without the G1 or G2 growth cycles of human somatic cells (Nishiyama et

al., 2006).

TRF2 (Telomeric Repeat binding Factor 2)

Like TRF1, TRF2 is a recruitment protein for the shelterin complex and a

negative regulator of telomere length. Like TRF1, TRF2 binds double-stranded DNA in


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vivo (Smogorzewska et al., 2000), recognizing the same TAGGG sequence as TRF1

(Hanaoka et al., 2005). The presence of TRF2 is dependent on telomere length. TRF2

also functions as a telomerase regulator, preventing telomerase access to the telomere

when the tract is long. Overexpression of TRF2 causes telomere shortening

(Smogorzewska et al., 2000). Unlike TRF1,Xenopus TRF2 has been found to associate

with telomeric chromatin throughout the cell cycle (Nishiyama et al., 2006).

TRF2 also binds DNA as a homodimer. It also shares the TRFH-hinge-

SANT/Myb domain structure of TRF1, with a basic Gly/Arg-rich domain at the N-

terminus instead of the acidic domain of TRF1 (de Lange, 2005; Palm and de Lange,

2008). However, TRF1 and TRF2 do not heterodimerize (Broccoli et al., 1997). The

TRFH site of TRF2 interacts with the shelterin accessory factor Apollo (Chen et al.,

2008).

As discussed above, TRF2 is implicated in the formation of t-loops. The

involvement of the tumor suppressor protein p53 at the t-loop junction with TRF2 is

notable given the interaction of these proteins in the DNA damage response pathway

(Palm and de Lange, 2008). A dominant-negative mutant of TRF2 lacking the basic and

myb domains removed endogenous TRF2 from telomeres and caused telomere end-to-

end fusions (van Steensel et al., 1998). Deletion and dominant-negative mutation of

TRF2 are lethal because they activate the p53/p21 DNA damage response pathway,

mediated by ATM as the signal transducer (Karlseder et al., 1999). The role of TRF2 in

averting the action of the ATM kinase may be either through maintaining the higher-

order telomere structure (for example, t-loops) or through abrogating a more downstream

step in the response pathway (Palm and de Lange, 2008). TRF2 binds ATM in vitro and

inhibits the autophosphorylation of ATM on S1981, providing one possible mechanism

for preventing the response of the ATM kinase to DNA damage (Karlseder et al., 2004).

POT1 (Protection of Telomeres 1)

POT1 is a negative regulator of telomerase activity, inhibiting the enzyme by

restricting access to the 3 priming single-stranded overhang (Kelleher et al., 2005; Xin et

al., 2007). Thus, the telomerase-regulating activity of POT1 is dependent upon its ability

to bind single-stranded telomeric DNA (Kelleher et al., 2005). POT1 is characterized by

several OB (oligonucleotide/ oligosaccharide/ oligopeptide binding) folds, which are also

common to many other single-stranded DNA binding proteins (Kerr et al., 2003). POT1

binds single-stranded DNA and negatively impacts telomerase activity in vitro (Kelleher

et al., 2005). The ability of POT1 to regulate telomere length requires the interaction

with both single-stranded telomeric DNA and TPP1 (Kelleher et al., 2005; Hockemeyer


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et al., 2007). RNA interference of TPP1 function or a point mutation in the TPP1-

binding domain of POT1 impedes POT1 localization to telomeres (Liu et al., 2004;

Hockemeyer et al., 2007).

POT1 is implicated in the regulation of the terminal specificity of telomeres, as

POT1 knockdown cells show randomization of the typically conserved ATC-5 terminal

nucleotide sequence. Inhibition of POT1 causes chromosome ends to behave as sites of

DNA damage (Hockemeyer et al., 2005). POT1 is the crucial player in the repression of

the ATR pathway at telomeres, and a deficiency in POT1 initiates the DNA damage

response (Wu et al., 2006). This repression capability is also dependent upon the

association of POT1 with TPP1. POT1 mutants without the TPP1 interaction domain are

not recruited to the nucleus (Palm and de Lange, 2008).

TPP1 (TINT1 / PTOP / PIP1)

TPP1, named for its former identities as TINT1, PTOP, and PIP1, is another

component of the shelterin complex (Palm and de Lange, 2008). This protein bridges

TIN2 with POT1 through its C-terminal TIN2 interaction domain and its central POT1

interaction domain (Figure 1.3). As mentioned above, this linkage is crucial for the

recruitment of POT1 to telomeres, as the deletion or mutagenesis of TPP1 results in

POT1 depletion from telomeres (Liu et al., 2004). Impaired function of TPP1 results in

deprotection of telomeres and subsequent lengthening consistent with POT1 loss

phenotypes. TPP1 is also necessary for the nuclear localization of POT1 (Palm and de

Lange, 2008).In its interaction with POT1, TPP1 plays a critical role in capping the ends of

telomeres by protecting the single-stranded overhang. The N-terminal OB fold of TPP1

interacts with telomerase, providing a physical association between telomerase and

potentially recruiting it to the telosome. The recruitment and regulation of telomerase by

TPP1 provides a balance to the negative regulation performed by the capping function of

the TPP1-POT1 complex (Xin et al., 2007). TPP1 is also critical to the interaction of

TRF1 and TRF2 through TIN2 by regulating the connectivity of TRF1 and TRF2

subcomplexes and directly regulating the TRF1-TIN2 interaction (O'Connor et al., 2006).

TIN2 (TRF1-Interacting Nuclear protein 2)

TIN2 is a negative regulator of telomere length due to its interaction with TRF1

(Kim et al., 1999). TIN2 protects TRF1 from poly(ADP-ribosyl)ation in vitro and

theoretically from subsequent removal from telomeres by tankyrase (Ye and Lange,

2004). Diminished TIN2 function endangers the end-capping function of TRF1, resulting


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in activation of the ATR damage response pathways at telomeres (Palm and de Lange,

2008).

The TRFH dimerization domain of TRF1 interacts with a FxLxP motif in the C-

terminal region of TIN2, identified by crystal structure. In contrast, Far-Western assays

and a gel-filtration chromatographic analysis identified a C-terminal motif of TRF2 and

the N-terminal residues of TIN2 that mediate the interaction between those two proteins

(Chen et al., 2008). Because these two interactions each depend on different domains,

they can take place concurrently, allowing TIN2 the ability to bridge TRF1 and TRF2

and stabilize the complex (Ye et al., 2004). TIN2 also recruits TPP1 to shelterin via a

distinct N-terminal protein interaction domain (Palm and de Lange, 2008). This TPP1

binding is necessary for effective TRF2-TIN2-TRF1 interaction. The TIN2-TPP1

interaction appears to be a crucial factor in overall shelterin stability (O'Connor et al.,

2006).

RAP1 (Repressor/Activator Protein 1)

The human ortholog of the yeast Repressor/Activator Protein 1 (RAP1) is

recruited to telomeres via its interaction with TRF2. RAP1 has a BRCT motif at its N-

terminus, a Myb domain that may interact with another unknown protein, and a C-

terminal RCT domain (Li et al., 2000). This C-terminal region interacts with the hinge

domain of TRF2. Yeast RAP1 binds to telomeric DNA via a second Myb domain, but

mammalian RAP1 lacks this direct binding capability. Mammalian RAP1 is therefore

dependent upon TRF2 for its interaction with the telosome complex (Palm and de Lange,2008). Rap1 is a negative regulator of telomerase length, though the exact mechanism

for its regulation is unknown (De Boeck et al., 2009).

PinX1 is a TRF1-interacting telomerase inhibitor

PinX1 is a 328 amino acid, 45kDa protein that interacts with TRF1 and inhibits

telomerase in vivo and in vitro. As described in detail above, human telomerase is

composed of the catalytic subunit, hTERT, and the template RNA, hTR. The PinX1 N-

terminal (aa 1-142) and C-terminal (aa 254-328) domains both interact with hTERT.However, only the C-terminal region appears to be responsible for telomerase inhibition,

and so it is designated the telomerase inhibitory domain (TID). Overexpression of the

PinX1 C-terminal TID substantially reduces the replicative capacity of a cell population

by hindering the telomere-lengthening function of telomerase. Depletion of endogenous

PinX1 by RNA interference causes an increase in the enzymatic activity of telomerase


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and a lengthening of telomeres, as shown by a standard TRAP assay (Zhou and Lu,

2001).

PinX1 binds to the region of hTERT that also interacts with the hTR template

RNA. The PinX1-interacting hTERT fragments (aa 17-546 and aa 523-924) overlap with

the hTERT N-terminal RNA-binding domain (aa 326-620). The mutation or absence of

the hTR RNA subunit did not reduce the PinX1-hTERT interaction effect (Banik and

Counter, 2004).

The C-terminal (aa 253-328) region of PinX1 also binds directly to the telomerase

hTR subunit in vitro. The N-terminal (aa 2-252) region of PinX1 is unable to bind hTR.

The binding of PinX1 to hTR is thus not attributable to the general RNA binding ability

of PinX1 (Banik and Counter, 2004) via its G-patch, a glycine-rich domain widely

conserved among eukaryotes (Aravind and Koonin, 1999).

PinX1 also binds hTR in vivo in telomerase-positive cells, but not without the

presence of hTERT. This is important to note considering the overlaps of the hTR and

PinX1 binding regions of hTERT. Still, hTERT immunoprecipitates with hTR in the

presence of PinX1, implying that even though the hTERT binding regions for PinX1 and

hTR overlap, binding of PinX1 does not obstruct the interaction of the telomerase

components in vivo. Rather, it is likely that PinX1 accomplishes its function as a

telomerase inhibitor via its interaction with the assembled hTERT-hTR holoenzyme.

Telomerase activity is repressed in vivo by the PinX1 TID binding to the assembled

hTERT-hTR complex (Banik and Counter, 2004).

Both human and yeast PinX1 homologues are nucleolar proteins (Lin et al.,

2007). Yeast PinX1p (also known as Gno1p) has functions in both telomerase regulation

and snRNA/rRNA processing. The yeast PinX1p is involved in pre-ribosomal RNA

(rRNA) processing and end-trimming of two small nucleolar RNAs (snoRNAs). The G-

patch RNA binding domain is essential for both of these functions. Unlike human PinX1,

the yeast homologue is not known to interact with any other telomeric proteins. A study

by Guglielmi and Werner (2002) asserts that though it does have a function in telomere

length regulation, the yeast Gno1p does not act in vivo as a negative regulator of

telomerase activity like its human homologue. In fact, in contrast to the lengthening of

telomeres caused by depletion of hPinX1, depletion or mutation of the yeast Gno1p in

this study resulted in slightly shorter telomeres. However, this effect may be an artifact

of slow growth in the mutant phenotype (Guglielmi and Werner, 2002). This artifact

effect is more probable in light of a study overexpressing yeast PinX1 in wild-type cells

that found a consequent shortening of telomeres. Cell extracts with yPinX1

overexpression exhibited decreased telomerase activity when compared to those cells


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with vector-only control, showing that yeast PinX1 directly decreases telomerase

enzymatic activity in vitro. One resulting model proposes that yPinX1 homologue

regulates telomerase by sequestering the reverse-transcriptase protein subunit TERT

(Est2p in yeast) in a nucleolar inactive complex lacking the RNA component (Lin and

Blackburn, 2004).

The model for human regulation of telomerase is different. Overexpression of

hPinX1 does force hTERT to translocate from the nucleoplasm into the nucleolus.

However, the centrally located domain of hPinX1 that mediates nucleolar localization of

hTERT is functionally and structurally separated from the C-terminal TID. A substantial

fraction of cells coexpressing hTERT and the central region of the hPinX1 polypeptide

(aa 142-254) showed nucleolar concentration. Those cells coexpressing hTERT with

either the hPinX1 N-terminal (aa 1-142) or C-terminal terminal TID (aa 254-328) domain

showed no such effects on subnuclear localization patterns of hTERT. Lin et al. assert

that their results show that a double mutation affecting the nucleolar localization capacity

of PinX1 does not impact telomerase inhibition. TRAP assays comparing overexpression

of wild-type PinX1 and this mutated PinX1 with a vector control do not show dramatic

telomerase inhibition with either PinX1 as effectively as the authors claim (Lin et al.,

2007). Still, with this data on the overexpression of the central hTERT subnuclear

localization domain, along with the above identification of the TID by Zhou and Lu

(2001), it is safe to conclude that sub-nuclear shuttling and telomerase regulation are

directed by different domains of hPinX1. This stands in contrast to the yeast PinX1

homologue, which purportedly accomplishes telomerase regulation via the mechanism of

nucleolar sequestering.

The function of PinX1 as a nucleolar protein that regulates telomere length is

conserved in the rat, supporting the possibility for conservation of function among other

vertebrates. The nucleolar localization function of PinX1 is also conserved in the rat.

Cellular overexpression of rat PinX1 in mouse cells caused gradual telomere shortening,

but no observable impact on telomerase activity. This does not conclusively refute the

possibility for rat PinX1 inhibition of telomerase, however, as a recombinant protein may

be necessary for detection of this effect (Oh et al., 2007).

Xenopus laevis PinX1 has been previously cloned and its expression in various

tissues characterized. There is differential PinX1 expression amongX. laevis tissues,

exhibiting a negative relationship between PinX1 abundance and telomerase activity

(Gaubatz, 2007). There have been preliminary investigations into the function ofX.

laevis PinX1 as a telomerase inhibitor, though there is no conclusive evidence thus far

(Constant, 2009).


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The roles of telomeres, TRF1 and PinX1 in oncogenesis

Because of their role in maintaining chromosome integrity and facilitating cell

proliferation, defective telomeres play a crucial role in oncogenesis. The expression of

the human telomerase TERT component in fibroblasts results in telomere elongation.

Continuous activity of telomerase causes these cells to be immortal, avoiding senescence

caused by the gradual telomere shortening that occurs during successive cell replication

(Verdun and Kalseder, 2007). Telomerase-activity-linked expression of hTERT occurs in

about 85% of cancerous cells. Such expression is thus the best available marker for

cancer detection (De Boeck et al., 2009).

Because of their regulatory effects on telomerase, TRF1 and PinX1 have

implications in the proliferative potential of cancerous cells. Overexpression of the TRF1

isoform Pin2 induces apoptosis in cells with short telomeres, though it does not have this

affect in cells with long telomeres. Down-regulation of TRF1/Pin2 permits themaintenance of long telomeres by allowing greater telomerase access to the chromosome

ends. TRF1/Pin2 abundance is significantly lower in cancerous tissues, suggesting that this

down-regulation is key for the proliferative capability of tumor cells (Kishi et al., 2001a).

Overexpression of human PinX1 decreases telomerase activity and therefore also

reduces the proliferative potential of cancerous cells. Depletion of PinX1 expression

increases telomerase activity and tumorigenicity (Zhou and Lu, 2001). Overexpression

of human PinX1 forces hTERT to translocate from the nucleoplasm to the nucleolus of

cancer cells. A mutant version of hPinX1 reported in some cancers does not have the

capacity for this hTERT nucleolar localization. Mutations of two residues that are found

commonly in human hepatocarcinoma patients were introduced to an hPinX1 construct,

disrupting the ability of the mutant construct to accumulate in the nucleolus. This

blockade to nucleolar localization would, in turn, disrupt the ability of PinX1 to sequester

hTERT in the nucleolus (Lin et al., 2007). These observations suggest that PinX1 may be

a tumor suppressor in humans.

The interaction of PinX1 and Trf1

PinX1 was first identified as a TRF1/Pin2-interacting protein in a human HeLa

cell yeast two-hybrid cDNA library screen (Zhou and Lu, 2001). Pin2 is a splice variant

of TRF1, structurally identical apart from an internal 20 aa deletion. Pin2 is 5-10 fold

more abundant in cells than TRF1 (Shen et al., 1997).

The interaction of PinX1 and TRF1/Pin2, both in vivo and in vitro, was further

confirmed via coimmunoprecipitation and colocalization experiments (Zhou and Lu,


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2001). The TRFH domain of human TRF1/Pin2 binds the R284-E-G-R-D-F-T-L-K-P-K-

K-R-R-G-R299 fragment of PinX1. This interaction was confirmed by ITC (isothermal

titration microcalorimetry) using TRF2 as a non-binding control. This PinX1 binding

region contains the conserved motif F/Y-X-L-X-P, also found in the TRFH-interacting

domains of TIN2 and Apollo. The PinX1 R291-L and the TRF1 R142-F residues were

crucial to this interaction in mutagenesis studies (Chen et al., 2008).

Xenopus as a model organism

X. laevis is an especially ideal model for studying cellular and developmental

genetics, as its embryos are particularly large and manipulable. Progeny are quickly

generated from in vitro fertilizations and embryos have easily identifiable developmental

stages. These characteristics, along with differences in telomere length and patterns

between individuals, makeX. laevis an interesting model for the study of telomere

variation (Bassham et al., 1998).

In humans, telomerase is not expressed in most somatic tissues. This lack of

telomerase causes telomeres to shorten and cells to senesce after a certain quantity of

divisions (Chan and Blackburn, 2004). Xenopus laevis is an ideal model organism for the

study of telomere function because it exhibits constitutive telomerase activity in all

somatic tissues (Bousman et al., 2003) and developing embryos (Mantell and Greider,

1994). Xenopus telomere length regulation is different from that observed in humans, in

that telomeres inXenopus somatic cells are no shorter than germ cells (Bassham et al.,

1998). InXenopus egg extracts,the activity of telomerase is higher when associated with

interphase than with mitotic chromatin, suggesting that telomere length is actively

regulated by cell cycle dependent recruitment of TRF1 to telomeres (Nishiyama et al.,

2006).

As discussed in more detail above,Xenopus laevis TRF1 (xlTRF1) has previously

been sequenced and characterized, and a clone is readily available in the lab for further

manipulations. BothX. laevis andX. tropicalis have homologs of TRF1 with a truncated

D/E-rich acidic domain. The hTRF1 motif, RXXPDG, that interacts with the hTRF1

regulator tankyrase is not present in this shortened acidic domain, which may have

implications for xlTRF1 regulation. TheX. laevis dimerization domain shares 49%

identity and 68% similarity with the human equivalent (Crumet et al., 2006). This high

identity within theX. laevis TRFH dimerization domain may have implications for the

interaction of this region with PinX1.


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Xenopus laevis PinX1 (xlPinX1) has previously been cloned and is readily

available in the lab. A previous study found a negative relationship between endogenous

PinX1 abundance and telomerase activity inX. laevis tissues (Gaubatz, 2007). The

differential telomerase activity among tissues is not due only to diffusible inhibitors of

telomerase, as shown by mixing experiments combining extracts from tissues with high

and low telomerase activity (Bousman et al., 2003). The TERT unit ofX. laevis

telomerase has been cloned (Kuramoto et al., 2001) and characterized previously.

Human PinX1 interacts with the hTERT regions aa 17-546 and aa 523-925 (Banik and

Counter, 2004).

Experimental goals and design

This study strives to investigate the in vitro interaction of TRF1 and PinX1 in

Xenopus laevis. This interaction has previously been established with the human

homologues of these proteins (Zhou and Lu, 2001). Ascertaining whether theXenopus

proteins interact in a similar fashion will provide another building block towards using

Xenopus as a model organism for the study of telomeres and telomeric regulation. This

study aims to use epitope tagged clones of the xlTRF1 and xlPinX1 proteins to

investigate interaction in a coimmunoprecipitation assay. As it has not yet been

confirmed inX. laevis that TRF1 does in fact form a homodimer, the experiment will also

test this potential in parallel, through coimmunoprecipitation assays with epitope-tagged

full-length xlTRF1 and the xlTRF1 dimerization domain.

One possible hypothesis is that theX. laevis proteins TRF1 and PinX1 will exhibit

the same interaction as they do in humans. Xenopus TRF1 has exhibited very similar

structure and functions to those of humans and other vertebrates (Crumet et al., 2006;

Nishiyama et al., 2006). If theX. laevis proteins are found to interact, this outcome

would be in line with the trend of intra-species conservation of telomeric protein function

among vertebrates.

Another possible hypothesis is thatX. laevis PinX1 and TRF1 will be shown not

to interact. Such a finding would move toward a model forX. laevis telomere regulation

that is, at least in one aspect, substantially different from that for humans. Finding that

the proteins do not interact would imply the evolution of some other mechanism by

which X. laevis regulates its abundant telomerase.

This hypothesis can first be refined by probing the conservation of the interacting

domains of PinX1 and TRF1 betweenX. laevis and humans. The amount of identity in

these regions between species (discussed further in the results and discussion section


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below) could provide a refined hypothesis of the likelihood that PinX1 and TRF1

interact. But if the domains are not highly similar between species and the proteins are

still shown to interact, this finding would provide valuable information as well in the

quest for better understanding of the relationships of protein interactions among and

within species.


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Materials & Methods

Sequence alignment analysis

Preliminary analyses were performed to visually compare TRF1 and PinX1 gene

and protein sequences between different species. Sequence data was retrieved from the

NCBI Entrez Nucleotide database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=

nuccore) and aligned using the program Multiple Sequence Alignment by ClustalW

(http://align.genome.jp/). The program BoxShade (http://www.ch.embnet.org/software/

BOX_ form.html) was then used to shade the sequences in a visually logical pattern

corresponding to similarity and identity of individual residues. The PinX1 protein

sequences used were human (AAS19507),Xenopus laevis (sequence of clone available in

lab, Gaubatz, 2007),Xenopus tropicalis (translation from mRNA EU520259), mouse(NP_082504) and rat (ABO28828). The TRF1 sequences used were human

(AAB54036),Xenopus laevis (sequence of clone available in lab, Crumet and Shampay,

unpublished),Xenopus tropicalis (NP_001137394), mouse (NP_033378), and chicken

(ACD68268).

PCR construction of Myc-tagged xlTRF1dd

Primer design

Polymerase Chain Reaction (PCR) primers were designed to amplify the gene of

interest with epitope tag sequences (Table 2.1). The primers were designed using gene

sequences of an xlTRF1 (Crumet et al., 2006) clone already available. The full-length

xlTRF1 protein sequence of the clone of study is available in Figure 3.1. The program

DNA Strider (CEA, France) was used to reverse translate epitope tag sequences into DNA

primer sequence. Primers were designed using the program AmplifX v.1.4 (Institut Jean

Roche) to check for potential primer-dimer complications and to anticipate annealing

temperatures for PCRs. Primers for xlTRF1dd sequences were obtained from IDT.

Table 2.1. PCR primers used to create Myc-tagged xlTRF1dd construct.

Primer name 5 3 Sequence

xlTRF1dd-F (w/ MluI site,

kozak sequence, Myc tag)

ACGCGTaccatgggcGAACAGAAGTTGATTTCCGAAG

AAGACCTCgatgacacggccGCTGTTG

xlTRF1dd-R (w/ NotI site) GCGGCCGCttaCTGAATATCCAGTTCTTCTTTTGCT


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Amplification of desired construct

PCR was used to amplify the dimerization domain from the xlTRF1 T16

pCR2.1 plasmid clone available (Crumet et al., 2006). Each 25 L PCR was run in a

thin-walled PCR-specific 0.2 mL tube. PCRs included the following reagents, each an

aliquot from a master mix designed for multiple reactions: 0.25 L Phusion High-

Fidelity DNA Polymerase (2 U/L; Finnzymes), 5 L 5X Phusion High-Fidelity buffer

(1X final; Finnzymes), 0.5 L dNTPs (200 M final each of dATP, dGTP, dCTP, dTTP;

New England Biolabs), 0.25 L each of forward and reverse primers (0.25 M final;

IDT), 100 pg T16 xlTRF1 pCR2.1 plasmid DNA template (generous gift of J.

Shampay), and 18.25 L nanopure H2O to bring to final volume. PCRs were performed

in a MJ PTC-200 Peltier Gradient Thermal Cycler (MJ Research).

Conditions for the PCR program were optimized by first running several reactions

over a gradient of annealing temperatures to find the optimal temperatures of 50C (first 5cycles) and 64C (final 25 cycles) for xlTRF1dd amplification. Reactions were run under

cycle conditions with two different annealing temperatures, as specified in Table 2.2.

Such optimization was crucial due to the particularly lengthy nature of the xlTRF1dd-F

primer.

Table 2.2. Cycling program for amplification of xlTRF1dd.

Step # Purpose Temperature Duration

1 Initial Denaturation 98C 30 sec

2 Denaturation 98C 10 sec

3 Annealing 50-58C gradient 30 sec

4 Extension 72C 20 sec

5 Repeat steps 2-4 (5 cycles total)

6 Denaturation 98C 10 sec

7 Annealing 64-72C gradient 30 sec

8 Extension 72C 20 sec

9 Repeat steps 6-8 (25 cycles total)

10 Final Extension 72C 10 min

11 Hold 4C


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To ensure the production of appropriately sized products, a portion of each PCR

product was analyzed on 1% SeaKem LE agarose gel in 1X TBE buffer (ISC). Gels were

run at 106 V for approximately 45 minutes. A 100 bp DNA ladder (New England

Biolabs) was used as a size marker for product comparison. Gels were stained for 15-30

minutes in ethidium bromide (1 g/mL) and de-stained for 10-20 minutes in deionized

water. Gels were visualized via trans-UV illumination in a Kodak EDAS 290.

Subcloning of xlTRF1dd PCR product into intermediate vector

PCR product purification

PCR products were purified to stop the action of the Phusion polymerase in

preparation for topoisomerase-mediated subcloning of the xlTRF1dd gene. The products

from multiple PCRs were pooled in one vial. Nanopure water was added to bring thetotal volume to 100 L. An equal volume (100 L) of phenol:chloroform:IAA was

added to the vial and the reaction was shaken until cloudy. The sample was then spun for

10 minutes at 2000 xg and the aqueous phase was transferred to a clean tube. To this

aqueous phase, 1/10 volume (10 L) of 3 M NaCl was added, followed by 2.5 volumes

(250 L) of cold (-20C) 95% EtOH. The reaction was vortexed briefly, then placed on

ice for 20 minutes. The tube was centrifuged at 12000 xg for 15 minutes. Excess EtOH

was removed by pipetting, leaving a pellet of DNA in the bottom of the vial. The pellet

was dried at room temperature for about 20 minutes before proceeding with the next step.

The dried DNA pellet was resuspended in 21.5 L of nanopure H2O with 2.5 L

of 10X Taq buffer (New England Biolabs) and 0.5 L Taq polymerase (New England

Biolabs) to add an A overhang for TOPO cloning. The reaction was incubated for 10

minutes at 72 C before proceeding immediately to TOPO cloning. Products from the

Taq polymerase A overhang addition were confirmed by visualization on a 1.0% agarose

gel.

Subcloning and transformation ofE. coli

Subcloning of xlTRF1dd was performed using the TOPO TA Cloning kit

(Invitrogen). Four microliters of purified PCR product with A overhangs was combined

with 1L TOPO kit salt solution and 1L pCR8/GW/TOPO vector (hereafter

abbreviated TOPO vector). The reaction was mixed gently and incubated for 5 minutes

at room temperature and then placed on ice.

These clones were then transformed into One Shot chemically competentE. coli

cells (Invitrogen). For each reaction, one vial of One Shot cells was thawed on ice and


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divided into two 25 L aliquots. Subsequently, 1.5 L of the TOPO cloning reaction

product were added to one of these 25 L aliquots of cells. The other aliquot was used as

a positive control for transformation by addition of 10 pg of pUC19 vector, a supercoiled

control plasmid. Cells were mixed gently with these vector additions and incubated on

ice for 30 minutes. Cells were heat shocked for 30 seconds in a 42C water bath and

immediately returned to ice. Next, 250 L of room temperature S.O.C. medium (2%

tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4,

20 mM glucose) was added to these cells. The vials were capped and shaken horizontally

at 200 rpm in a 37C incubator for one hour.

Reactions utilizing the xlTRF1dd transformants in the pCR8/GW/TOPO

vector were plated on LB agar plates containing 100 g/mL spectinomycin.

Transformants with the pUC19 control plasmid were selected for on 100g/mL

ampicillin LB agar plates. Each reaction was plated in two different amounts of 10 L

and 290 L to ensure at least one plate with a good distribution of colonies. Plates were

incubated overnight at 37C.

Small plasmid DNA isolations

Plasmid-containing bacteria were grown overnight in 2 mL LB liquid cultures

inoculated with appropriate selection antibiotic, shaking at 200 rpm in a 37C inclubator,

slanted for aeration. From each of these cultures, 500 L were transferred into plastic

reaction tubes and centrifuged at 12,000 xg for 1 minute. The supernatant was removed

and the cells were resuspended in 100 L STET buffer (5% Triton X-100, 8% w/vsucrose, 50 mM EDTA, 50 mM Tris (pH 8.0)). Following suspension, 10 L of freshly-

prepared lysozyme solution (10 mg/mL) was added to each sample. Samples were boiled

for 45 seconds then immediately centrifuged for 10 minutes at 12,000 g. The resulting

large, balloon-like pellets of cellular detritus and chromosomal DNA were then removed

from the samples and discarded. To the remaining supernatant, 100 L of cold

isopropanol was added. The samples were then vortexed and chilled at -20C for 30

minutes. DNA was collected by centrifugation for 10 minutes at 12,000 g. The

supernatant was removed from each sample and the remaining pellets were resuspended

in 25 L RNase A solution (50 g/mL in TE buffer). Samples were incubated for 30

minutes at 37C, and stored at 4C for later use.

Analysis of isolated plasmid DNA

TOPO plasmid isolations were checked for proper size of insert with double

digests with MluI and NotI restriction enzymes. DNA was treated with a master mix (1x


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final concentration NEBuffer 3, 0.1 mg/mL final concentration bovine serum albumen)

and cut with five units (0.5 L each) of MluI and/or NotI enzymes (New England

Biolabs). Reactions were incubated overnight at 37C. Restriction digest products were

visualized by electrophoresis in 1.0% SeaKem LE agarose in 1X TBE buffer.

Samples of plasmid DNA were purified by polyethylene glycol (PEG)

precipitation. An equal volume of 13% PEG in1.6 M NaCl was added to plasmid DNA

preparations. The reaction was placed on ice for 20 minutes, then centrifuged at

12,000xg at 4C for 15 minutes. The resulting pellet was rinsed with cold 70% EtOH and

suspended in 20 L nanopure H2O. DNA concentration was quantified on 0.8% SeaKem

LE agarose in 1X TBE buffer by comparison with lanes of-phage DNA cut with

HindIII (25ng and 75ng; New England Biolabs). Sequencing was performed following

procedure outlined below.

Construction of pTNT in vitro translation clone

Ligation into pTNT vector

Proper clones containing both the xlTRF1dd and Myc-tag epitope sequences were

cut out of the TOPO vector using the same restriction digestion protocol outlined

above. The pTNT in vitro translation vector (Promega) was cut in its multiple cloning

site using these same MluI and NotI restriction enzymes, using the procedure described

above for TOPO clone analysis. DNA was treated with a master mix (1x final

concentration NEBuffer 3, 0.1 mg/mL final concentration bovine serum albumen) and cut

with five units (0.5 L each) of MluI and/or NotI enzymes (New England Biolabs). The

samples were then heat inactivated for 20 minutes at 65C. Samples of each of the

digestion products (3 L each of MluI- and NotI-digested pTNT vector and TRF1dd-

TOPO vector) were run on a 0.8% agarose gel and quantified by comparison with lanes

of-phage DNA cut with HindIII (25ng and 75ng).

The xlTRF1dd-Myc clones were then ligated into the pTNT in vitro translation

vector, using an approximately 2-to-1 ratio of insert to vector to produce optimal ligation

results. The reaction combined 12 L double-digested TRF1dd-TOPO plasmid DNA(approximately 150-200 ng), 2 L double-digested pTNT vector (approximately 65

ng), 2.5 L T4 DNA ligase buffer with 10mM ATP (1X final; New England Biolabs), 1

L T4 NA ligase (400 units/L; New England Biolabs), and nanopure H2O to a final

volume of 25 L. The ligation reaction was incubated overnight at 17.5C.


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Transformation ofE. coliwith pTNT-xlTRF1dd vector

The ligation product was transformed into One Shot chemically competentE.

coli cells (Invitrogen). One vial of One Shot cells was thawed on ice and divided into

two 25 L aliquots. Subsequently, 3.5 L of the pTNT-xlTRF1dd ligation product were

added to one of these 25 L aliquots of cells. A positive control reaction and

transformation procedures were performed as outlined above forE. coli transformation

with TOPO clones. The reactions were then plated on LB agar plates with 100 g/mL

ampicillin. The pTNT-xlTRF1dd transformation reaction was plated in three different

amounts of 10 L, 25 L and 240 L. Control pUC19 transformations were plated with

10 L and 265 L. Plates were incubated overnight at 37C. Plasmid DNA was isolated

and analyzed by restriction digestion.

pTNT-xlTRF1dd plasmid purification

The pTNT-TRF1dd plasmid was purified using the QIAfilter Midi Plasmid

Purification Kit (all components from Qiagen unless otherwise noted). A large 100 mL

of ampicillin-selective (100 g/mL) LB medium culture was inoculated with 200 L of

starter culture from the initial ligation products. The culture was grown overnight at 37C

shaking at 300 rpm. The culture was divided into two 50 mL conical tubes and

centrifuged at 3000xg at 4C for 30 minutes. One of the resulting pellets was

resuspended by vortexing in 4 mL Buffer P1 with RNase A (final 100 g/mL). Next, 4

mL of Buffer P2 was added and the solution was mixed by inversion before incubation at

room temperature for 5 minutes. To the lysate was added 4 mL chilled Buffer P3. The

lysate was mixed by inversion, poured into the QIAfilter cartridge, and incubated at room

temperature for 10 minutes. A QIAGEN-tip 100 was equilibrated by gravity flow of 4

mL of Buffer QBT through the tip. The QIAfilter cartridge outlet nozzle tip was

removed, the plunger was inserted, and the lysate was filtered into the equilibrated tip.

The lysate was allowed to enter the tip resin by gravity, then the tip was washed with two

10 mL volumes of Buffer QC. The DNA was eluted into a glass centrifuge tube with 5

mL Buffer QF. The eluate was precipitated with 3.5 mL of room temperature

isopropanol, mixed, and centrifuged at 15,000xg for 30 minutes at 4C. The supernatant

was decanted and the pellet was washed with 2 mL of room temperature 70% EtOH. The

solution was centrifuged at 15,000xg for 10 minutes at 4C. The supernatant was

decanted and the pellet dried for 10 minutes. The pellet was redissolved in 100 L

nuclease-free TE.

A sample of this DNA (0.5 L) was digested with 4 units each of MluI and NotI

restriction endonucleases (New England Biolabs) in NEBuffer3 and BSA (1X final each;


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New England Biolabs) in a final volume of 20 L. These samples of the purified DNA

(0.2 L, 0.5 L, and 1.0 L uncut, and 1L and 3L restriction digested) were run on a

0.8% SeaKem LE agarose gel in TBE and quantified by comparison with lanes of-

phage DNA cut with HindIII (25ng and 75ng). Sequencing was performed following the

procedure below.

Sequence analysis of plasmid DNA

For sequencing of both TOPO and pTNT clones, approximately 150-300 ng

of purified template DNA were combined with either 3.2 pmol of the appropriate forward

or reverse primer to a final volume of 9 L in 0.2 mL PCR tubes. These samples were

sent to the Vollum DNA sequencing core facility. The resulting sequence files were

visualized with FinchTV software (Geospiza,

http://www.geospiza.com/Products/finchtv.shtml). Text files of each of the sequences

were created in DNA Strider (CEA, France). These text files were then aligned in the

DNA anti-parallel method and analyzed for inconsistencies between forward and reverse

sequences. The resulting sequence data was compared with published sequences to

ensure the construct was properly inserted without mutations or deletions.

In vitro expression of proteins

Protein products were created using the TNT coupled reticulocyte

transcription/translation kit (all reagents from Promega except plasmid templates). The

xlTRF1-Flag plasmid template was the generous gift of J. Jin (cloning described in Jin,

2009). The TNT T7 quick master mix was rapidly thawed then placed immediately on

ice. The reaction was assembled with the following components: 20 L TNT T7 quick

master mi

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