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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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Odd S. Gabrielsen
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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MBV4230
Odd S. Gabrielsen
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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1. TAFs as coactivators
TATA
Promoter
TBP
TF
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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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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Odd S. Gabrielsen
Multiple complexes contain TAFs
Red common to all
Dark blue only in TFIID
and TFTC, but not SAGA
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Odd S. Gabrielsen
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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Odd S. Gabrielsen
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.
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2. Mediator
TATA
Promoter
TBP
TF
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MBV4230
Odd S. Gabrielsen
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.
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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?