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Transcription in Eukaryotes I and II - University of Floridaoge.med.ufl.edu/courses/gms...

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Transcription in Eukaryotes I and II Jörg Bungert, PhD 352-273-8098 [email protected]
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Transcription in Eukaryotes I and II

Jörg Bungert, PhD

352-273-8098

[email protected]

Objectives

• Know the basal promoter elements and the basal transcription factors.

• Know the RNA polymerase II CTD phosphorylation cycle.

• Understand reporter-gene assays.

• Know and understand enhancer function.

• Understand Chromosome Conformation Capture (3C).

• Know the structure and function of the mediator.

• Know histone modifications and how they impact gene expression.

• Know the different chromatin modifying activities (HAT, HDAC, HMT, chromatin remodeling complexes).

• Know what a DNAseI hypersensitive (HS) site is.

• Know RNA polymerase I and III transcription complex formation.

Reading: Lodish 6th edition, chapter 7 (pp. 276-317).

Transcription in Eukaryotes - Overview

Eukaryotic RNA Polymerases

1. Pol I - Transcribes rRNA genes (28S, 18S, 5.8S RNA genes); accounts for 80-90% of total

cellular RNA mass

2. Pol II – Transcribes protein-encoding mRNA’s, and several non-protein encoding genes;

consists of 12 subunits that are conserved among diverse eukaryotic organisms

3. Pol III - Transcribes tRNA, 5S RNA, 7S RNA (component of signal recognition particle genes,

and other small RNA-encoding genes (including genes encoding RNA’s involved in splicing;

tRNA and 5S genes have internal promoter (i.e., within body of gene)

4. Each RNA Pol can be distinguished by differences in sensitivity to α-amanitin

5. Each RNA Pol has multiple subunits, is similar from yeast to mammals, and is more complex

than the E. coli RNA polymerase

6. Some subunits of each RNA Pol share some sequence similarity to E. coli RNA pol, and all

three eukaryotic RNA Polymerases share come common subunits

7. The C-terminal end of the largest subunit of pol II subunit contains a repeated 7 a.a. motif

1. This repeated region is termed the C-terminal domain (CTD)

2. Active transcription is correlated with phosphorylation of CTD

3. CTD is associated with several RNA processing factors

8. All three RNA Pols use TBP (TATA binding protein) in assembly of pre-initiation complex

9. New report (2005) of previously unknown mitochondrial RNA pol (spRNAP-IV) transcribing

some nuclear mRNA-encoding genes with promoters different from those using pol II

Subunit Structure of RNA Polymerases

The TATA-Box

Basal Promoter Elements in Higher Eukaryotes

Housekeeping, constitutively

expressed, genes are transcribed from

multiple start sites. Tissue-specific

and regulated genes are transcribed

from a single start site. The

mechanism of dispersed transcription

initiation is not well characterized.

Focused transcription initiation takes

place at genes harboring specific basal

promoter elements (e.g. TATA, INR,

DPE). TATA and DPE are present in

about 15 to 20% of genes transcribed

by Pol II, the INR is found in about

50% of the genes.

Juven-Gershon and Tjian, Developmental Biology, 2009

RNA Polymerase II Transcription

The pre-initiation complex (PIC):

PIC assembles at the basal promoter region of a gene

PIC consists of RNA pol II, 5 general transcription factors (GTF’s), and promoter DNA

GTF’s = TFII D, TFIIB, TFIIF, TFIIE, TFIIH (and TFIIA?)

Identified by in vitro

transcription assay (Run

Off) using as a DNA

template the adenovirus

major late promoter

(Ad2MLP).

(e.g., HeLa cells)

TATA-INR-DPEE-Box

Steps in Transcription Complex Formation

TBP binds to the

minor groove

and bends the DNA.

Bending may facilitate

melting of the DNA

or assembly of the

transcription complex.

The binding of TBP is

facilitated by TFIIAIIA

Steps in Transcription Complex Formation

TFIIB bridges the TBP-TATA

box complex with the RNA

polymerase.

Steps in Transcription Complex Formation

TFIIF interacts with the RNA

Polymerase and is required

for preventing unspecific

initiation at non-promoter

sites as well as for efficient

transcription elongation.

Steps in Transcription Complex Formation

TFIIE stimulates the

helicase and ATPase

activities of TFIIH

Steps in Transcription Complex Formation

TFIIH is a multi-protein

complex that contains

kinase, helicase, and

ATPase activity. It is

required for melting

the DNA during initiation

and for the phosphorylation

of serine 5 of the CTD.

Steps in Transcription Complex Formation

“closed” complex

“open” complex

TBP/TATA box complex and TFIID

TFII D composed of::

•TBP – TATA-Binding Protein

•TAF’s – TBP-associated factors

Also, evidence for:

- Cell type-specific TBP’s

- TAF complexes w/out TBP that can functionally

replace TFIID

Structure of the Preinitiation Complex

TBP/TFIIA/TATA-Box

TBP/TFIIA/TFIIB/TATA-Box

Structure of the Elongating RNA Polymerase

Cheung et al., EMBO J. 2011

Red: Antibody for

phosphorylated CTD

Green: Antibody for un-

phosphorylated CTD

74/EF/75b: ecdysone induced

high level transcription in

“puff” regions.

CTD Heptapeptide: Thr Ser Pro Thr Ser Pro Ser (yeast, 26X)

Tyr Ser Pro Thr Ser Pro Ser (mammals 52X)

Phosphorylation of RNA Pol II CTD Correlates with

Gene Activation

Transcriptional pause is a frequent event

during elongation caused by slight

misalignment of the RNA 3’OH and the

active site. It is self-reversible and

regulated by numerous cellular factors.

Transcriptionally arrested complexes, in

which the RNA 3’OH and active site are

irreversibly misaligned, resume elongation

after Pol II mediated transcript cleavage

and realignment of the active site and the

RNA 3’OH end, processes that are

stimulated by TFIIS.

Transcription Elongation Factors

CTD phosphorylation cycle: TFIIH subunit Kin 28 phosphorylates Ser 5. Transcription

elongation complex (TEC) is arrested at checkpoint for pre-mRNA capping. P-TEFb

(Bur1/2 or Ctk1 in yeast) then phosphorylates Ser 2, which allows further elongation.

Specific phosphatases (possibly Ssu72 and FCP1, which are required for Pol II

Recycling) dephosphorylate the CTD at Ser 5 and Ser2.

Modification of RNA Pol II CTD During

Transcription Elongation

Activation of Transcription

Tissue-specific Gene Expression

Simple gene expression array analysis.

cDNAs are spotted onto nitrocellulose

or nylon membranes and hybridized to

radio-labeled RNA from liver, kidney, or brain.

For identifying tissue-specific DNA control elements

the genomic regions of the liver-specific genes

can be isolated from genomic libraries. The

upstream promoter regions can then be ligated in

front of reporter genes and analyzed for tissue-

specific expression.

Identification of Regulatory DNA Elements by

Progressive Deletions and Reporter Gene Assays

Assume TTR is a liver specific gene identified in

the previous experiment. The upstream regulatory

region of this gene was cloned in front of a reporter gene

(Reporter Gene: e.g. LacZ, luciferase, GFP).

The reporter gene constructs were transiently

transfected into a liver cell line.

The data demonstrate that a tissue-specific

cis-regulatory DNA element, required for activated

expression of the reporter gene is located between

the 5’end of fragment 2 and the 5’end of fragment 3.

In addition, a basal promoter element is located

between the 5’end of fragment 4 and the

5’ end of fragment 5.

The regulatory DNA element that is located

between the 5’end of fragment 2 and the 5’end of

fragment 3 can be further analyzed for the

presence of transcription factor binding sites.

A. In silico analysis for known factors using databases.

B. Electrophoretic Mobility Shift Assays (EMSA).

C. DNA Footprinting.

D. Chromatin Immunoprecipitation.

Modular Structure of Transcription factors

-Plus: Nuclear Localization Sequences (NLS), Protein/Protein

Interaction Domains, Sites of Modification.

Identification of Transcription Regulators by

Reporter-Gene Assays

The previous experiment identified

a cis-regulatory element of the

TTR gene that mediates high-level

expression in a liver cell line. To

identify a transcription factor that

binds to the cis-regulatory element

(X) and activates transcription, co-

transfection experiments can be

performed. Plasmid 1 carries the

gene encoding the transcription

factor of interest, while plasmid 2

carries regulatory sequence X

linked to a reporter gene. A control

experiment would be performed

with plasmid 2 lacking sequence X.

Identification of Transcription Regulators by

Reporter-Gene Assays

Identification of DNA binding and Transcription

Regulation Domains

Genome Wide Analysis of Protein Chromatin Interactions Using

ChIP-seq or ChIP-chip

Farnham P.J., Nature Rev. Genet., 2009

Hormone mediated transfer of GR to the Nucleus

Enhancer and Promoter of a Liver-Specific Gene

Cooperative Binding of Transcription Factors to DNA

Enhanceosome

•Enhancer can be located upstream or downstream of genes (or in introns)

•Enhancer act in an orientation independent manner.

•Many Enhancer elements act in a tissue-specific manner.

•The size of enhancers is 200 to 400bps.

•They act by a tracking, looping, or linking mechanism.

The drosophila eve gene is regulated by

many enhancer elements that confer

Expression of the eve gene in specific

stripes of the developing embryo. The

stripe 2 enhancer is composed of binding

sites for activating proteins (Bicoid: Bcd,

Hunchback, Hb) and repressing proteins

(Kreisler, Kr, and Giant, Gnt). During

early embryonic development, a gradient of

transcription factors is set up in the

multi-nucleated embryo (syncytium). At

this stage, the concentration of bicoid and

hunchback is high in stripe 2, whereas the

concentration of kreisler and giant

is low.

Activation of the stripe 2 enhancer by a gradient of transcription

factors in the developing Drosophila embryo

Ptashne and Gann, 2002

Possible Mechanisms of Enhancer Function

Dean, A., Trends in Genetics, 2006

Model of dynamic associations

of genes with transcription factories.

Chromatin loops (black) extruding

from chromosome territories (gray).

Transcribed genes (white) in RNA

Pol II factories (black circles).

Potentiated genes (free loops) that

are not associated with Pol II factories

are temporarily not transcribed and can

migrate to a limited number of pre-

assembled Pol II factories (dotted arrows)

Osborne et al., Nat. Genet., 2004Szentimayr and Sawadogo, Nucl. Acids Res.. 2002

Transcription and Transcription Factories

Dekker, Nat. Methods 2006

Chromosome conformation capture

Active genes dynamically colocalize to shared sites

of ongoing transcription

Actively transcribed genes associate

with RNA Pol II foci. (a) RNA immuno-

FISH of Hbb-b1 transcription (red) with

RNA Pol II staining (green) in erythroid

cells. (b) DNA immuno staining of

Eraf (red) and RNA Poll II (green).

(c) Comparison of the percentage

of alleles exhibiting a gene transcription

signal by RNA FISH (black), with the

Percentage of loci that overlap with an

RNA Pol II focus by DNA FISH.

3C showing interactions between

b-globin LCR and the transcribed

Eraf and Uros genes in erythroid cells.

Calr is a ligation and PCR control.

E: erythroid

B: brain cells

nuclei were fixed for 5

Minutes (in a) or for 10 min (in b)

Osborne et al., Nat. Genet., 2004

Model of a transcription factory (diameter

70 nm) containing 8 polymerases (green

crescents). Genes are reeled through these

factories (one polymerase per gene).

Heat shock gene activation and formation

of a transcription factory that facilitates

reinitiation.

Transcription Factories

Sutherland, H., and W.A. Bickmore, Nat. Rev. Genet., 2009

Activation of Transcription via Mediator

Structure of Mediator

Transcription Repressors Recruit Chromatin Modifying

Protein Complexes

Silencing of Chromatin Domains by

Polycomb Repressor Complexes

The PRC complex mediates the silencing of genomic loci. It is recruited to specific sites in chromatin by

the PRC responsive elements (PREs) and proteins that interact with PREs. There are two PRC complexes.

PRC2 methylates H3K27 and represses gene expression. PRC1 recognizes methylated H3K27 and

methylates CpGs. This leads to a stably repressed chromatin configuration that can be transmitted to

daughter cells after mitosis.

Transcription Activators Recruit Chromatin Modifying

Protein Complexes

Histone Modifications In Euchromatin and Heterochromatin “Histone Code”

A histone mark

associated with

sites of Pol II

recruitment is

trimethylated

H3K4. This

modification has

been shown to

assist in the

recruitment of

TFIID.

Dimethylated H3K4

is associated with

active chromatin

domains

H3K27 and H3K9

trimethylation is

associated with

repressed

chromatin.

H3K36 methylation

is associated with

transcription

elongation.

Histone modifications

Histone acetylation• Occurs on lysine side chains of N-terminal tails of histones

• Changes charge of histone tails – makes them less basic (neutralizes positive charge)

• May weaken histone-DNA interactions and “open” the nucleosome

• May alter histone-histone interactions

• May alter interactions between histones and regulatory proteins

• May facilitate binding of regulatory proteins to cis-acting elements in DNA

• Effects may be on individual nucleosomes and/or higher order chromatin structure

• Makes histone tails more -helical

• Catalyzed by histone acetyltransferases (HAT’s)

• Reversible by histone deacetylases (HDAC’s)

Histone methylation• Does not alter net charge of histone tails

• Mono-, di-, tri-methylation of lysine side chain

• Catalyzed by histone methyltransferases (HMT’s)

• Methylation of histone tails thought to be stable

• Histone demethylase isolated in 2005 – LSD1, lysine-specific demethylase, removes a

methyl group from one particular residue (H3, K4), probably others

ATP-Dependent Chromatin Remodeling Complexes

Steps in Gene Regulation

Genes are normally embedded in inaccessible

chromatin. The first step in the activation of

gene expression is often the recruitment of

chromatin remodeling complexes (Swi/SNF) by

pioneer transcription factors that recognize

their respective binding site in the context of

nucleosomes or higher order structure. In the

example shown here, the yeast DNA binding

factor SWI5 recruits the SWI/SNF complex to

its binding site in chromatin. In an ATP

hydrolysis dependent manner Swi/SNF

mobilizes nucleosomes, rendering a short

segment of chromatin accessible (nuclease

sensitive). Subsequently, SWI5 and other

DNA binding proteins recruit histone

acetyltransferases (HATs) like yeast GCN5.

Steps in Gene Regulation

HATs acetylate H3 and H4 N-terminal tails and

provide docking sites for other co-regulatory

protein complexes. For example, acetylated

histones recruit additional HATs that acetylate

neighboring nucleosomes leading to the

spreading of accessible chromatin.

Steps in Gene Regulation

The partial opening of chromatin structure allows other DNA binding transcription

factors to access their binding sites.

Steps in Gene Regulation

The binding of additional transcription factors is followed by

recruitment of mediator…

Steps in Gene Regulation

… and the transcription preinitiation complex. This leads to a segment of DNA that is completely devoid of a

nucleosome, thus forming a DNAseI hypersensitive site. The example shown above is just one way genes can

be activated.

In other cases, like heat shock genes, the transcription complex is already bound at the promoter but stalled.

Gene activation leads to a change in the transcription complex that renders it elongation competent.

Recent evidence suggests that highly expressed genes are recruited to transcription factories in the nucleus.

Transcription factories are domains in the nucleus that contain multiple aggregated transcription complexes.

Active Genes are Associated with Accessible Chromatin

Mapping DNase I Hypersensitive Sites

DHS1 DHS2

B

15kb

B

3.1kb

1.5kb

Exon 1Probe

DHS1

DHS2

[ DNase I ] 0

- 15 kb

- 3.1 kb

- 1.5 kb

Southern blot

1) Treat chromatin/cells with DNase I

2) Purify genomic DNA

3) Digest DNA with

restriction enzyme (BamHI)

4) Fractionate DNA on agarose gel

5) Blot gel to membrane (Southern blot)

6) Hybridize blot with probe

7) Expose gel to film for autoradiogram

RNA Polymerase I Transcription

RNA Polymerase III Transcription

U6 Gene


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