An Integrated View to Gene ExpressionAn Integrated View to Gene Expression

From Orphanides & Reinberg, Cell, 2002From Orphanides & Reinberg, Cell, 2002

Biochemistry of Transcription and ChromatinBiochemistry of Transcription and Chromatin

In essence, gene transcription is a simply In essence, gene transcription is a simply process, that can be divided into individual stepsprocess, that can be divided into individual steps

Margaritis, Holstege, Cell 2008

.

““All information to correctly express a All information to correctly express a protein-encoding gene is in its sequence”protein-encoding gene is in its sequence”

boundary element

matrixattachment

region

enhancers

-4000

locus control region

boundary element

matrixattachment

region

-500 -40

distal promoter core promoter

Gene X

enhancer

promoter

What is the “gene expression” parts list for a simple What is the “gene expression” parts list for a simple eukaryote?eukaryote?

A simple eukaryote: the yeast Saccharomyces cerevisiae

6200 genes (5% are spliced), non-coding transcripts but no microRNAs

± 250 gene-specific transcription factors± 150 chromatin remodeling and modifying factors

~65 general transcription machinery proteins~20 transcription elongation proteins

± 800 “other” factors involved in regulation:such as 117 protein kinases, ±200 components of the ubiquitin

machinery, mRNA splicing proteins, mRNA export factors

What is the “gene expression” parts list for a complex What is the “gene expression” parts list for a complex eukaryote?eukaryote?

A more complex eukaryote, Homo sapiens:22,000 mRNA genes (95% are spliced), ~1,000 microRNAs,

5,000-10,000 long non-coding RNAs

~2,500 gene-specific transcription factors~1,000 chromatin proteins

~75 general transcription machinery proteins~50 transcription elongation proteins

“other” factors involved in regulation:such as ~500 protein kinases, ±1200 components of the

ubiquitin machinery, mRNA splicing proteins, mRNA export factors

NF-B TATA Interleukin 2

“upstream activator” “basal machinery”

gene-specific general or basal

promoter-distal core promoter

-40 +40anywhere

p65p50

“Cofactors”

Gene-specific transcription factors Gene-specific transcription factors require “Cofactors” (activators or require “Cofactors” (activators or repressors) to mediate repressors) to mediate their regulatory effectstheir regulatory effects

MediatorChromatin modifiers, e.g. HATs,

HDACs, PRMTs, KMTs, KDMsChromatin remodellers, e.g. ATP-dependent

enzymes, nuclear laminaHistone binders, e.g. histone chaperones,

HP1, elongation factors19S proteasomal particle

.

““All information to correctly express a All information to correctly express a protein-encoding gene is in its sequence”protein-encoding gene is in its sequence”

boundary element

matrixattachment

region

enhancers

-4000

locus control region

boundary element

matrixattachment

region

-500 -40

distal promoter core promoter

Gene X

but .......but .......it is very difficult to find functional elementsit is very difficult to find functional elements

by DNA sequence gazingby DNA sequence gazing

enhancer

promoter

Accessibility Promoter (~50 bp) recognition

The example of human chromosome 22400 core promoters= 400 x 50 bp= 2,000 bp

2,000 in 48,000,000 bp1 to 24,000 windows of 50 bp

The example of yeast6,200 core promoters= 310,000 bp

310,000 in 12,400,000 bp1 to 40 windows of 50 bp

How to locate a core promoter?How to locate a core promoter?

Sandelin et al 2007 Nature Reviews Genet. 8, 424

Location of core promoter by genomic sequencing of mRNA endsLocation of core promoter by genomic sequencing of mRNA endsSharp or focused pol II promoters:

10-20% of totalcontain a TATA-box

Broad type of pol II promoters:50-70% of total

reside in a CpG islands

General classification ofof pol II promoters

Sandelin et al Nat Rev Genet 8 (2007); Tora and Timmers TiBS 35 (2010)

TFIID binds both modified chromatin and promoter DNATFIID binds both modified chromatin and promoter DNA

DNA contacts: TBP TATATAF1/2 INRTAF6/9 DPE

Protein contacts:TAF1 H3-K9,14ac; H4-KacTAF3 H3-K4me3

Jacobson et al. Science 288 (2000)Vermeulen et al. Cell 131 (2007)

Guenther et al. Cell 130 (2007)

Five distinct DNA sequences in focused core promotersFive distinct DNA sequences in focused core promoterscan attract the basal machinerycan attract the basal machinery

TATA INR

“basal machinery”

DPE

-30/-24 -2/+5 +28/+32

MTE

+18/+27

... but they are functionally redundant... but they are functionally redundantand are present in a small set of promoters.....and are present in a small set of promoters.....

TATAWAAR YYANWYY RGWYVCSARCSS

BRE

SSRCGCC

-38/-32

““Back to the seventies”Back to the seventies” Paradigm for mRNA synthesis: bacterial enzyme (4 subunits) plus sigma-factor Three nuclear RNA polymerases in human cells Recombinant DNA cloning technology Characterization of DNA viruses for animal cells (SV40, polyoma, adenovirus)

Identification of the basal pol II machinery Identification of the basal pol II machinery

[C][C]

HeLa WCEHeLa WCE

phosphocellulose

0.04/0.10.04/0.1 0.35/0.30.35/0.3 1.0/0.851.0/0.850.6/0.50.6/0.5

[A][A] [B][B] [D][D]

TFIIBTFIIBTFIIETFIIETFIIFTFIIFTFIIHTFIIH

TFIIDTFIID(B-TFIID)(B-TFIID)

““all basal factors and pol II subunits are encoded all basal factors and pol II subunits are encoded by highly-conserved and unique genes”by highly-conserved and unique genes”

Identification of the basal (or general) transcription factors Identification of the basal (or general) transcription factors by complementation in an by complementation in an in vitroin vitro pol II transcription assay pol II transcription assay

using the TATA promoter of the adenovirus ML geneusing the TATA promoter of the adenovirus ML gene

TFIIATFIIA

33 basal factor polypeptides33 basal factor polypeptides13 RNA pol II peptides (+)13 RNA pol II peptides (+)

46 polypeptides46 polypeptides

Basal transcription factors assemble in a stepwise Basal transcription factors assemble in a stepwise fashion fashion

B74F E

34

56

H

pol II

TBP TATA

30

-40 -30 -20 -10 +1 +10

Promoter Promoter recognitionrecognition

TBP-TFIIDTBP-TFIIDTFIIBTFIIBTFIIFTFIIF

Promoter Promoter openingopening

TFIIETFIIETFIIHTFIIH

AbortiveAbortiveInitiationInitiation

ProductivProductivee

InitiationInitiationTFIIHTFIIH (TFIIH)(TFIIH)

Core promoter elements are bound by basal transcription factorsCore promoter elements are bound by basal transcription factors

TATA INR

“basal machinery”

DPE

-30/-24 -2/+5 +28/+32

MTE

+18/+27

TATAWAAR YYANWYY RGWYVCSARCSS

BRE

SSRCGCC

-38/-32

TFIIB TFIID

.

TBP/TFIID:TBP/TFIID:

38-kDa in humans; 27-kDa in yeast TATA-binding protein (TBP) is the major sequence-specific DNA binding

component of TFIID TBP is (always) associated in large multi-protein complexes two complexes, TFIID and B-TFIID, support pol II transcription 13 TBP-associated factors (TAFs) are in TFIID and B-TFIID has only one TAF DNA binding by TBP (and TFIID) is slow and has limited sequence selectivity TBP consists of a divergent N-terminus and a highly conserved C-terminal

domain of 180 aa (almost) all functions reside in this conserved domain TBP is sufficient for basal transcription in vitro activated transcription in vitro requires TAFs as cofactors TAFs have been found to interact with many transcription factors TAFs are not essential for transcription activation in vivo

Properties of TBP and TFIID

TAFI-41

TBP

TBP

TAFI-48 TAFI-110

TBPBRF1

B-TFIID SL-1

TFIIIB

pol III

BDP

pol I

pol II

pol II

TFIID

13 TAFs

BTAF1/Mot1p

TBP

TBP

Nucleolus

Peri-nucleolar

Nucleoplasm

TBP

TBP distribution in human cell lines

TAFI-63

TBP

TBP-TAF complexesTBP-TAF complexes

• TFIID TFIID 13 TAFs (250-18 kDa) plus TBPTBP pol IIpol II

• B-TFIIDB-TFIID BTAF1(Mot1p) plus TBPTBP pol II

• SL 1 SL 1 4 TAFs (110, 63, 48, 41) plus TBPTBP pol I

• TFIIIBTFIIIB 2 TAFs (150, 90 kDa) plus TBPTBP pol III

Proteins directly affecting basal transcription by pol IIProteins directly affecting basal transcription by pol II

TBP-associated factors:

TAF1 can inhibit TATA-bindingrecognizes INR-elementHAT activity ?double bromo-domain binding acetylated nucleosomes

TAF2 recognizes INR-elementTAF3 recognizes trimethylated lysine-4 of histone H3

involved in muscle differentiation (?)TAF4b selective for TGF signalingTAF6/9 contacts DPEBTAF1/MOT1p dissociates TBP-NC2-TATA complex dependent on ATP

relaxed DNA binding specificityNC2 blocks TFIIA/TFIIB entry to preinitiation complex,

induces TBP sliding on DNArelaxed DNA binding specificity

TFIIA prevents action of repressors like NC2increased stability of DNA interaction

TATA GeneX

“basal machinery”

Time for a coffeecoffee break

.

RNA polymerase II: consists of 12 subunits; all but one (RPB4) are essential RBP1 and RBP2 are homologous to ’ and of RNAP some subunits (RBP5, -6, -10, -10) shared with pol I and pol III

RPB1 ’RBP2 RBP3 ””RBP4RBP5RBP6RBP7RBP8RBP9RBP10RBP10RBP11

Nobel Prize Chemistry 2006: Kornberg"for his studies of the molecular basis of eukaryotic transcription"Speaker CHAINS 28/11/2011, Maarssen

.

RNA polymerase II:

has little intrinsic proofreading activity low elongation efficiency, but high processivity promoter-specific initiation requires ATP hydrolysis RBP1 subunit contains unique heptad repeat structure (YSPTSPS)

forming the so-called CTD (52 repeats in mammals) CTD is target for kinases and phosphatases CTD of elongating pol II is hyper phosphorylated transcription cycle involves CTD-phosphorylation cycle truncation of CTD by 50% gives conditional phenotypes in yeast CTD is involved in many protein interactions

(mRNA processing factors, coactivators)

.

Cramer, Bushnell and Kornberg (2001) Science 292, 1863

.

Gnatt et al. (2001) Science 292, 1876

.

RNA polymerase II:

has little intrinsic proofreading activity low elongation efficiency, but high processivity promoter-specific initiation requires ATP hydrolysis CTD is target for kinases and phosphatases CTD of elongating pol II is hyper phosphorylated transcription cycle involves CTD-phosphorylation cycle truncation of CTD by 50% gives conditional phenotypes in yeast CTD is involved in many protein interactions

(mRNA processing factors, coactivators)

The CTD is differentially phosphorylated during transcriptionThe CTD is differentially phosphorylated during transcription

CTD-kinases: cdk7/cyclinH of TFIIH (Ser5/7) cdk8/cyclinC of Mediator (Ser5) cdk9/cyclinT of PTEFb (Ser2) CTK1 (Ser2)

CTD-phosphatases:FCP1 (Ser2)

Ssu72 (Ser5)

Ser2-P

Ser5-PSer7-P

consensus CTD-repeat: YSPTSPS

.

RNA polymerase II:

has little intrinsic proofreading activity low elongation efficiency, but high processivity promoter-specific initiation requires ATP hydrolysis CTD is target for kinases and phosphatases CTD of elongating pol II is hyper phosphorylated transcription cycle involves CTD-phosphorylation cycle truncation of CTD by 50% gives conditional phenotypes in yeast CTD is involved in many protein interactions

(mRNA processing factors, coactivators)

Discovery of cofactors

mRNA processing: cappingsplicingpoly(A)-tail addition

The phosphorylated CTD attractsattracts mRNA processing factors

.

RNA polymerase II:

has little intrinsic proofreading activity low elongation efficiency, but high processivity promoter-specific initiation requires ATP hydrolysis CTD is target for kinases and phosphatases CTD of elongating pol II is hyper phosphorylated transcription cycle involves CTD-phosphorylation cycle truncation of CTD by 50% gives conditional phenotypes in yeast CTD is involved in many protein interactions

(mRNA processing factors, coactivators)

Discovery of cofactors

SRB proteins were identified by genetic means in SRB proteins were identified by genetic means in yeastyeast

the CTD of yeast pol II carries 26 heptad repeats

deletion to less than 12 repeats gives rise to conditional phenotypes (inositol-

auxotrophy, cold-sensitivity)

yeast cells with less than 10 repeats are non-viable

mutations in SRB (Suppressor of RNA polymerase B ) genes restore viability

biochemical purification showed that SRB proteins are associated in a multi-

subunit complex termed the Mediator, which can bind directly to pol II

Mediator also known as ARC, SMMC, NAT, PC2, DRIP, TRAP complexMediator also known as ARC, SMMC, NAT, PC2, DRIP, TRAP complex

The Mediator complexThe Mediator complex

Conserved from yeast to mammalsAssociated with RNA pol II

Required for transcription nearly all protein-coding genesRequired for response to activators

Unified nomenclature for the Mediator complexUnified nomenclature for the Mediator complex

Mediator has at least two functions:required for the response to activators

stimulates basal txpn levels

Malik and Roeder (2005) Trends Biochem. Sci. 30, 256

Mediator mechanismMediator mechanism

Negative components

Enzymatic activities

Opposite effects on genes upon deleting different components?

Signal transduction?

Transcription of HIV is controlled at the level of elongationTranscription of HIV is controlled at the level of elongation

10-20% of genes contain paused pol II around 20-40 bp of TSS10-20% of genes contain paused pol II around 20-40 bp of TSS

Transcription elongation factors enhance Transcription elongation factors enhance the processivity of RNA polymerase IIthe processivity of RNA polymerase II

TFIIS re-activates paused pol II by inducing exonucleolytic cleavage Elongin (trimeric complex of elongin A , B and C) decreases Km ELL TFIIF FACT (facilitates chromatin transcription) dissociates

nucleosomes upon transcription DSIF (DRB sensitivity inducing factor) counteracts NELF

(negative elongation factor)-pausing of pol II P-TEFb (cdk9/cyclinT kinase) phosphorylates the CTD on Ser2 PAF1 complex Spt6 helps transcription through chromatin by binding dissociated H3 ....

The curious case of divergent transcriptionThe curious case of divergent transcription

.

Concepts in transcription regulation:Concepts in transcription regulation:

transcription factor accessibility and recruitment are important regulatory mechanisms

basal transcription factors are non-redundant and non-modular and they function position- and orientation-dependent

gene-specific factors are (often) redundant and dimeric and they function relatively position- and orientation-independent

gene-specific factors and cofactors are part of multi-gene families and they contain separate modules for DNA- or factor-binding and for transcription regulation

chromatin-dependent cofactors are large protein complexes

chromatin-dependent complexes can have both positive and negative effects on transcription

synergism, protein-modification and abundance are important regulatory mechanisms for gene-specific factors