TAFs and the Mediator - Universitetet i oslo

TAFs and the Mediator - Universitetet i oslo

Coactivators TAFs and the Mediators TF TBP TATA Promoter MBV4230 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. In vivo: OFF basal trx.app. TBP upstream transactivator ON basal trx.app. TFIIB TFIIA In vitro: ON +

TFIIE TFIIF TFIIH No activator response . Something missing MBV4230 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 transactivator

basalt trx.app. Activators (ordinary TFs) dont affect the basal trx.apparatus directly, but indirectly through coactivators and chromatin MBV4230 The coactivator bridges Roeder, R.G. (2005) Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Lett, 579, 909915. MBV4230 Coactivators = molecular bridges + chromatin remodeling TFs does not affect the basal transcriptional apparatus directly, coactivator but indirectly through coactivators upstream transactivator basal trx.app. Bridge

Chromatin remodelling MBV4230 3 main types of general coactivators 1. TAFs 2. Mediator/SRB-complex (holoenzyme components) TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBP

Multiple complexes that contain TAFs RNAPII- associated factors 3. General cofactors Non-associated factors 1. TAFs as coactivators TF TBP TATA Promoter MBV4230 1. Coactivators associated with TBP: TAFs TAFs = TBP associated factors

Function in activator response TAFs - Tjians biochemical studies 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 MBV4230 Identification of coactivators biochemical approach A280 Result: ? Basal trx.activity requires: core RNAPII + GTFs

Activator-responsive trx. activity requires : core RNAPII + GTFs + Coactivator MBV4230 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 Structure EM shows three to four major domains or lobes joined by narrower bridges, organized in a horseshoelike structure around a central channel. Two configurations observed: open and closed MBV4230

Conserved TAFs New nomenclature TAF1 = TAFII250 etc MBV4230 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: upstream transaktivator a TF recruits TFIID to the promoter through specific TAD-TAF contacts and this stimulates PICassembly basal trx.app. MBV4230 Multiple contacts to activators synergy 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 synergy Tr.respons linear A B A+B MBV4230 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 TAFs upstream transactivator basal trx.app. MBV4230

Functions of the TAF-complex 2. main function : TAFs bind corepromoter 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 MBV4230 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? MBV4230

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. MBV4230 More histone-like pairs MBV4230 TAF-model MBV4230

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 proteinprotein packing MBV4230 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 MBV4230

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) Substrates: see figure In yeast: kinase domains in two separate proteins 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 MBV4230 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 MBV4230 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 chromatinpackaged promoters MBV4230 Summary of TAF functions 2.

6. 5. 7. 4. 3. 1. 2. MBV4230 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). MBV4230

Sequential action 1. Recruitment by bound activators MBV4230 Sequential action 2. Nucleosome and core promoter recognition and binding MBV4230 Sequential action 3. Chromatin dynamics MBV4230 Sequential action

4. Initiation and elongation of transcription MBV4230 The TAF-complex in vivo: Hot debate on the importance of TFIID from general coactivator to gene-specific core-factor TAF-coactivator-model under scrutiny 1. interaction with activators - not verified in vivo

TAFs = biochemical artefacts or central actors in the activator response? 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 Only in vitro evidence Physiologically relevant? upstream transaktivator ? Importance supported by in vivo evidence TAFs

! basalt tr.app. MBV4230 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 MBV4230 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. SAGA

chromatin-remodeling complex Mot1 Repressor that binds TBPcomplex NC2 Global 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

TFTC TBP-free TAFII-containing complex MBV4230 Multiple complexes contain TAFs Red common to all Dark blue only in TFIID and TFTC, but not SAGA

MBV4230 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. SAGA chromatin-remodeling complex Mot1 Repressor that binds TBPcomplex NC2 Global 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 TFTC TBP-free TAFII-containing complex MBV4230 Multiple complexes with TBP

10x more TBP in a cell than there is of each of TAFs, SAGA, Mot1, NC2 and Nots MBV4230 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 TF TBP TATA Promoter MBV4230 3 main types of general coactivators 1. TAFs

2. Mediator/SRB-complex (holoenzyme components) TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBP Multiple complexes that contain TAFs RNAPII- associated factors 3. General cofactors Non-associated factors MBV4230 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 MBV4230 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 MBV4230

The holoenzyme Holoenzyme a challenge to the linear assembly of PIC A pre-assembled unit Quantitative estimates argues against that all RNAPII is in the form of holoenzyme MBV4230 Holoenzyme composed of several subcomplexes Srb 2,4,5 and 6 subcomplex Dominant suppressors Srb 4+6 essential for most promoters Srb 4 direct target for GAL4

Srb7 Med-proteins Essential, highly conserved from yeast to humans Associate with Srb 2-4-5-6 subcomplex through Srb4-Med6 contact Repressor complex several repressor-like proteins: Gal11, Sin4, Rgr1, Rox3 and Pgd/Hrs1/Med3 Mutations of these components may derepress subsets of genes 10-fold

Kinase subcomplex (Srb10 CDK) Recessive suppressors: Srb8, 9, 10 and 11 Negative role Srb10+11 forms a cyclin-CDK pair that phosphorylates Ser5 in the CTD tail Srb10+11 can phosphorylate free RNAPII MBV4230 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 proteins Isolated as a coactivator for thyroid receptor (TR) DRIP - vitamin D receptor-interacting proteins Isolated as a coactivator for vitamin-D receptor (VDR) Composition very similar to TRAP ARC - activator-recruited cofactor Isolated as a coactivator for SREBP-1a and Sp1, also coactivator for VP16, NFkB Identical with DRIP Human Mediator

Isolated as an E1A-interacting multicomplex with 30 polypeptides that bind activator-domains in E1A and VP16 CRSP, NAT and SMCC Contains several of the same subunits MBV4230 Conservation and variability Evolutionary conservation limited to a subset of mediator subunits Probably different variant forms of Mediator

MBV4230 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 MBV4230

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-CCDK8 pair (homologues of yeast Srb10+11) A smaller 500-700 kDa complex termed PC2/CRSP Lacks the cyclin-CCDK8 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

MBV4230 The yeast mediator model of activator-dependent transcription Different mediator proteins seem to have activatorspecific 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 MBV4230 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 Some evidence suggests that mediator functions in the reinitation step of the transcription cycle

More complex than suggested by the holoenzyme model 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 MBV4230 Mediator structure MBV4230 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. MBV4230 Model for mediator function

Promoter architecture mediator conformation Different conformations influence the re-entry of RNA polymerase II Particular combinations of activators influence the conformation of mediator. 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 MBV4230 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 TF TBP TATA Promoter MBV4230 3 main types of general coactivators 1. TAFs

2. Mediator/SRB-complex (holoenzyme components) TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBP Multiple complexes that contain TAFs RNAPII- associated factors 3. General cofactors Non-associated factors MBV4230 3. General cofactors

Factors that leads to increased activator response, but that are not associated with GTF or RNAPII Derived from the famous USA-fraction USA: Upstream stimulatory activity Fractionation revealed multiple positive and negative cofactcors PC: positive kofaktorer

PC1, PC2, PC3, PC4, PC5, PC6, ACF, CofA, HMG2 NC: negative kofaktorer Dr1, Dr2 [andre: NC1, MOT1/ADI, NOT1-4, TUP1] MBV4230 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-strrelse implication: freely floating or anchored?

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