CoactivatorsTAFs and the Mediators

TATA

Promoter

TBP

TF

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Activation of basal transcription- the missing link? RNAPII + GTF correct trx initiation in vitro, but do

not respond to activators Basal trx probably not occurring in vivo, eukaryotic promoters has to be

activated by upstream trx factors What is missing to reconstitute activator-dependent trx in vitro?

The coactivator was proposed to bridge the activator and other components necessary for transcription.

upstream transactivatorbasal trx.app.

ON

No activator response…. Something missing

basal trx.app.

OFF TBP

TFIIB

TFIIA

TFIIE

TFIIF

TFIIH

+

In vivo:

In vitro: ON

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Activation of basal transcription

activator-dependent trx requires several additional actors: basalt trx.apparatus - RNAPII + GTFs Transactivators - sequence-specific DNA-binding transcription factors Coactivators Chromatin remodelling

coactivator

upstream transactivatorbasalt trx.app.

Activators (ordinary TFs) don’t affect the basal trx.apparatus directly, but indirectly through coactivators and chromatin

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The coactivator bridges

Roeder, R.G. (2005) Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Lett, 579, 909-915.

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Coactivators = molecular bridges + chromatin remodeling

”Bridge”

Chromatinremodelling

coactivator

upstream transactivatorbasal trx.app.

TFs does not affect the basal transcriptional apparatus directly,but indirectly through coactivators

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3 main types of general coactivators

1. TAFs TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBP Multiple complexes that contain TAFs

2. Mediator/SRB-complex (holoenzyme components) RNAPII- associated factors

3. General cofactors Non-associated factors

1. TAFs as coactivators

TATA

Promoter

TBP

TF

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1. Coactivators associated with TBP: TAFs

TAFs = “TBP associated factors” TAFs - Tjians biochemical studies

Function in activator response TFIID reconstituted from recombinant TAFs makes the basal transcription

apparatus responsive to activators (def. coactivator)

Distinct TAFs for each transcription system RNAPI: SL1 = TBP + TAFIs

RNAPII: TFIID = TBP + TAFIIs

RNAPIII: TFIIIB = TBP + TAFIIIs TAFs

TBP

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Multiple TAFs with multiple activities

Large complex with 8 - 12 subunits Ranging in size from 250 kDa to less than 20 kDa

Highly conserved proteins (Drosophila, humans, yeast)

Functions associated with subunits hTAFII250 - HMG-box, bromodomains, serine kinase, binds the TAF-complex to TBP dTAFII150 - binds INR + downstream (human: separate factor = CIF) hTAFII135 /dTAFII110 - contacts Q-rich TADs (absent in yeast) hTAFII95/ dTAFII80 - WD40 repeat hTAFII80 /dTAFII60 - histone H4 like - contacts acidic TADs hTAFII55 - binds multiple activators, including P-rich TADs hTAFII31 /dTAFII40 - histone H3 like - contacts acidic TADs hTAFII28 hTAFII20 - histone H2B like

StructureEM shows three to four major domains or lobes joined by narrower bridges, organized in a horseshoe-like structure around a central channel. Two configurations observed: open and closed

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ConservedTAFs

New nomenclature

TAF1 = TAFII250

etc

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upstream transaktivator

basal trx.app.

Specific functions of the TAF-complex

1. main function: interaction with activators Physical contact found between TAFs and specific activators TAF-activator contact: each type of activator contacts a

particular TAF dTAF40 and 60 -- VP16, p53 (acidic TAD) dTAF150 and 60 -- NTF-1 (Ile-rich TAD) dTAF110 -- Sp1 (Q-rich TAD) dTAF55 -- CTF (P-rich TAD)

Logic: a TF recruits TFIID to the promoter through specific TAD-TAF contacts and this stimulates PIC-assembly

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Multiple contacts to activators synergy

A B A+B

Tr.

resp

ons

synergy

linear

Multiple TAF interactions might explain synergy synergy = > additive (linear) transcriptional response When two or more TFs together result in higher levels of activation

than the sum of each factors individual contribution

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Functions of the TAF-complex

2. main function : TAFs bind core-promoter elements TATA: through TBP INR: dTAF150 specific interaction with the INR-motif

dTAF250 also implied alternative anchoring of TFIID to PIC TAFII250, together with TAFII150, mediates binding of TFIID to the Inr and

can support Inr-mediated transcription. +GTF-contact: TAF110 and TAF60 bind TFIIA and TFIIB

upstream transactivator basal trx.app.

TAFs

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Functions of the TAF-complex

2. main function : TAFs bind core-promoter elements TATA: through TBP INR: dTAF150 specific interaction with

the INR-motif alternative anchoring of TFIID to

PIC +GTF-contact: TAF110 and TAF60 bind

TFIIA and TFIIB

DPE recognized through dTAF60 and dTAF40

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TAFs with nucleosome structure?

Several subunits with histone-like elements hTAFII80 /dTAFII60/ yTAFII60 - histone H4 like

hTAFII31 /dTAFII40/ yTAFII17 - histone H3 like

hTAFII20 /dTAFII30/ yTAFII68 - histone H2B like

In addition: hTAFII18 and hTAFII28 classfied as histone-like

Octamer-like structure possible?

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Histone fold = dimerization motif

Histone fold frequently found in TAFs More than half (9 out of 14) of the yTAFIIs contain a histone fold

motif, and they specifically assemble into five histone-like pairs The histone fold is the fundamental interaction motif involved in

heterodimerization of the core histones, H4 and H3, and H2A and H2B.

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More histone-like pairs

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TAF-model

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TAFs with nucleosome structure?

3. Main function - changing promoter topology or simply compact dimerization Structuring element within the TAF complex? Replacing nucleosomes, with DNA wrapped around - to mark active genes in mitosis?? Counter argument - histones contact DNA through Args not conserved in TAFs Probably simply to facilitate compact and tight protein–protein packing

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The enzymatic functions of the TAF complex 4+5+6. main function: enzymatic catalysis 4. HAT-activity

histone acetyl transferase activity in TAFII250 conserved activity in yeast, drosophila, humans mapped to central region histone acetylation opens chromatin, important in gene activation (more later) GTF substrates: TAFII250 acetylates TFIIE and TFIIF In vivo substrates still open Seminar: TAF1 activates transcription by phosphorylation of serine 33 in

histone H2B

5. Protein kinase TAF250 has two kinase activities

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The versatile TAFII250

TAFII250 is a bipartite kinase One Ser/Thr-kinase in the N-terminus (NTK) Another Ser/Thr-kinase in the C-terminus (CTK)

In yeast: kinase domains in two separate proteins Substrates: see figure

Itself - autophosphorylation GTFs, in particular TFIIF

Kinase required in vivo

Homologs TAFII130 and

TAFII145 in yeast,

TAFII230 and TAFII250 in Drosophila,

TAFII250 and cell cycle gene 1 (CCG1) in mammals

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Recent novel functions: ubitiquination and binding acetylated histones

6. Function: TAFII250 = a histone-specific ubiquitin-activating /conjugating enzyme (ubac).

TAFII250 mediates monoubiquitination of histone H1

Monoubiquitination of histones has been correlated with activation of gene expression

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Promoter recognition through TAFs bromo domains

7. Function: Bromodomains TAFII250 contains two

tandem bromodomain modules that bind selectively to multiple acetylated histone H4 peptides.

Bromodomains may target TFIID to chromatin-packaged promoters

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Summary of TAF functions

1.

2.

3.

4.

5.

6.7.

2.

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Summary of TAF functions (Drosophila)

Core promoter recognition factors by binding to the Inr and DPE by TBP:TATA box interactions,

can orient TFIID on the DNA (single-sided arrows).

Certain TAFs also activator targets capable of binding to activation

domains in vitro (double-sided arrows).

Enzymytic activities TAFII250 has two enzymatic

activities, a kinase and an acetylase, that can modify proteins (squiggly arrows).

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Sequential action

1. Recruitment by bound activators

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Sequential action

2. Nucleosome and core promoter recognition and binding

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Sequential action

3. Chromatin dynamics

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Sequential action

4. Initiation and elongation of transcription

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upstream transaktivator basalt tr.app.

TAFs

? !

Only in vitro evidencePhysiologically relevant?

Importance supported by in vivo evidence

The TAF-complex in vivo: from general coactivator to gene-specific core-factor

TAF-coactivator-model under scrutiny TAFs = biochemical artefacts or central actors in the activator response?

1. interaction with activators - not verified in vivo TAFs never found in genetic screens in yeast Hypotheses on TAF function essentially based on in vitro studies (Tjian) coactivator-model implies that most genes require the TFIID complex.

2. interaction with core-promoter elements - supported by genome-wide analysis in yeast Chimeric promoters

Hot debate onthe importance of TFIID

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The yeast attack - TAFs not universal factors required at all promoters

TAFs genes knocked-out - no global effects? TAFs not universally acting Each TAF controls only a

subset of genes

Swap experiments suggest a role in core promoter recognition The specificity of TAFs

linked to recognition of core promoter

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SAGAchromatin-remodeling complex

Mot1Repressor that binds TBP-complex

NC2Global repressor that binds TBP (in absence of DNA)

Nots

SAGA (yeast)chromatin-remodeling complex that contains the histone-like yTAFII17, yTAFII60 and yTAFII68, and also yTAFII25 and yTAFII90.

STAGA (human)Human version of SAGA

PCAF (human)chromatin-remodeling complex with several histone-like TAFs

TFTCTBP-free TAFII-containing complex

TFIID not the only TAF-complex- Multiple complexes contain TAFs Presence of TAFII subunits not

restricted to the well-known TFIID complex. Some TAFs have been found in other complexes, the function of which remains to be determined.

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Multiple complexes contain TAFs

Red common to all

Dark blue only in TFIID

and TFTC, but not SAGA

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SAGAchromatin-remodeling complex

Mot1Repressor that binds TBP-complex

NC2Global repressor that binds TBP (in absence of DNA)

Nots

SAGA (yeast)chromatin-remodeling complex that contains the histone-like yTAFII17, yTAFII60 and yTAFII68, and also yTAFII25 and yTAFII90.

STAGA (human)Human version of SAGA

PCAF (human)chromatin-remodeling complex with several histone-like TAFs

TFTCTBP-free TAFII-containing complex

TFIID not the only TAF-complex- Multiple complexes contain TAFs Presence of TAFII subunits not

restricted to the well-known TFIID complex. Some TAFs have been found in other complexes, the function of which remains to be determined.

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Multiple complexes with TBP

10x more TBP in a cell than there is of each of TAFs, SAGA, Mot1, NC2 and Nots

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Many TBP-complexes - implications TBP plays a role beyond TAFs

Trx probably regulered by several different TBP-containing complexes

TAF-complexes not global coactivators, but specific for subsets of genes

Unexpected importance of negative control of TBP? Negative regulation of TBP so important that three different complexes

(all essial for viability), have evolved - all bindning TBP.

2. Mediator

TATA

Promoter

TBP

TF

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3 main types of general coactivators

1. TAFs TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBP Multiple complexes that contain TAFs

2. Mediator/SRB-complex (holoenzyme components) RNAPII- associated factors

3. General cofactors Non-associated factors

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Isolation of Mediator

Genetic screens (in yeast) for suppressors of truncations in the CTD of RNAPII Supressors of cold-sensitive -CTD mutant identified the SRBs (Suppressors of RNA polymerase B) components, which

reside in a 1-2 Mda complex

Isolated biochemically (several systems) activator-dependent in vitro assays

on the basis of its ability to stimulate activator-dependent trx in vitro immunopurification assays based activator affinity purification step

Based on physical interaction with various activators and the CTD of RNAPII

identified a variety of proteins, including Gal11, Srb proteins, Med proteins, and Rox3

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The Mediator/SRB-complex is RNAPII-associated

Genetic isolation of supressors of CTD-deletion mutants SRBs

Biochemical isolation of a 20 polypeptide complex with coactivator properties

Consensus: Holoenzym = Mediator + RNAPII

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Mammalian Mediator

Several coactivators for specific factors have turned out to be more general than first understood and are probably identical or variants of the Mediator-complex

TRAP - TR-associated proteinsIsolated as a coactivator for thyroid receptor (TR)

DRIP - vitamin D receptor-interacting proteinsIsolated as a coactivator for vitamin-D receptor (VDR)Composition very similar to TRAP

ARC - activator-recruited cofactorIsolated as a coactivator for SREBP-1a and Sp1, also coactivator for VP16, NFkBIdentical with DRIP

Human Mediator Isolated as an E1A-interacting multicomplex with 30 polypeptides that bind activator-domains in E1A and VP16

CRSP, NAT and SMCCContains several of the same subunits

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Conservation and variability Evolutionary

conservation limited to a subset of mediator subunits

Probably different variant forms of Mediator

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Mediator – new nomenclature

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Functions of the Mediator/SRB-complex Evidence for in vivo trx function of mediator

temp.sens. Mutation in SRB4: non-permissive temp all mRNA syntesis stops immediately

Mediator/SRBs like a control panel for trx Kinase, activator like protein [ GAL11], proteins with repressor function

(SIN4, RGR1) and other control proteins

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Two variants of human Mediator- the smaller is the active version Purification procedures identified two complexes A larger 2 MDa complex termed ARC-L

Identical to complexes designated TRAP, DRIP, ARC, SMCC or NAT Contains the cyclin-C–CDK8 pair (homologues of yeast Srb10+11)

A smaller 500-700 kDa complex termed PC2/CRSP Lacks the cyclin-C–CDK8 pair CRSP70 is present only in the CRSP complex

The larger complex appears to be transcriptionally inert, while the smaller CRSP complex is the active species on the promoter

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The yeast mediator model of activator-dependent transcription

Different mediator proteins seem to have activator-specific roles

Activator contact The three activators

(GCN4, VP16 and GAL4) are shown binding to their DNA sites and recruiting yeast mediator to the promoter via a physical interaction with a mediator module

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Different temporal orders of recruitment of mediator and RNAPII 1. mediator RNAPII initiation of trx. 2. Mediator + RNAPII trx initiated later 3. RNAPII mediator initiation of trx

More complex than suggested by the holoenzyme model

Some evidence suggests that mediator functions in the reinitation step of the transcription cycle a reinitiation intermediate/scaffold that contains TFIIA,TFIID, TFIIH, TFIIE,

and mediator can be isolated

Re-entry of RNAPII as rate-limiting The rate at which RNAPII gains access to the preformed ‘scaffold’ may become

the rate-limiting step

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Mediator structure

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Conformations of the mammalian mediator complexes - flexibility?

ARC-L and CRSP EM composites of the

ARC-L and CRSP complexes

different structural conformations adopted by CRSP when isolated via

affinity interactions with either the VP16 or SREBP activator.

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Model for mediator function

Promoter architecture mediator conformation Particular combinations of activators

influence the conformation of mediator.

Different conformations influence the re-entry of RNA polymerase II to the promoter to initiate subsequent

rounds of transcription. panel A - a mediator

conformation that only promotes the slow re-entry of RNAPII

panel B promotes a faster RNAPII re-entry

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Multiple pathway model for transcriptional activation Activation signals from

DNA-bound activators can be transduced to RNAPII through multiple coactivator complexes including TAF-containing

complexes (upper yellow arrow) and mediator-like complexes

( lower yellow arrow).

The relative contribution of each pathway to trx regulation is likely to be activator- and/or promoter-dependent.

3.General coactivators

TATA

Promoter

TBP

TF

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3 main types of general coactivators

1. TAFs TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBP Multiple complexes that contain TAFs

2. Mediator/SRB-complex (holoenzyme components) RNAPII- associated factors

3. General cofactors Factors that leads to increased activator response, but that are not

associated with GTF or RNAPII

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A “transcriptosome” ?

The number of components so large that a “transcriptosome” will have a size of the same order as a ribosome Core RNAPII- 12 polypeptider, ca. 500 kDa Mediator/SRBs - ca.20 polypeptider GTFs - 6 stk ca. 16 polypeptider TAFs ≥ 8 polypeptider SWI/SNF complexet - mange polypeptider, ca. 2000 kDa ialt >70 polypeptider ≈ ribosom-størrelse

implication: freely floating or anchored?