TAFs and the Mediator by ewghwehws

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									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.


                            basal trx.app.                                   basal trx.app.
In vivo:   OFF                       TBP
                                             upstream transactivator
                                                                       ON
                                    TFIIB
                                    TFIIA

In vitro: ON                    +
                                    TFIIE                  No activator response
                                    TFIIF
                                                           …. Something missing
                                    TFIIH
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
                                                                               basalt trx.app.
                                       upstream transactivator




     Activators (ordinary TFs) don’t 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, 909-
                                                                                                                                  915.
MBV4230


 Coactivators = molecular bridges
 + chromatin remodeling

TFs does not affect the basal transcriptional apparatus directly,
but indirectly through coactivators              coactivator
                                                                basal trx.app.
                            upstream transactivator




                                           ”Bridge”




                                                                    Chromatin
                                                                    remodelling
MBV4230



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

          TF




                  TBP

                   TATA

                   Promoter
    MBV4230

    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
    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 horseshoe-like
     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

   main function: interaction with
 1.
 activators
      Physical contact found between TAFs and specific activators
      TAF-activator contact: each type of activator contacts a particular
       TAF                                                            basal trx.app.

           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)                upstream transaktivator

   Logic:     a TF recruits TFIID to the promoter through
       specific TAD-TAF contacts and this stimulates PIC-
       assembly
    MBV4230

    Multiple contacts to activators
     synergy




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




                                                                             Tr.respons
       than the sum of each factors individual contribution


                                                                                          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
                                           basal trx.app.
upstream transactivator
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
         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 protein–protein 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                                  Homologs
     One Ser/Thr-kinase in the N-terminus (NTK)                      TAFII130 and
     Another Ser/Thr-kinase in the C-terminus (CTK)                   TAFII145 in yeast,
            In yeast: kinase domains in two separate proteins        TAFII230 and
       Substrates: see figure                                         TAFII250 in
            Itself - autophosphorylation                              Drosophila,
            GTFs, in particular TFIIF                                TAFII250 and cell

       Kinase required in vivo                                        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 chromatin-
        packaged promoters
MBV4230



Summary of TAF functions




                2.             6.
                                    7.
                     5.

                                           4.
           1.                            3.


                          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
                                                                           Hot debate on
The TAF-complex in vivo:                                             the importance of TFIID

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                                              Importance supported by
                                           Only in vitro evidence           in vivo evidence
                                          Physiologically relevant?
                                                                      TAFs

                                       upstream transaktivator   ?               !   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 (yeast)
                                             chromatin-remodeling complex that
                                             contains the histone-like yTAFII17,
SAGA                                         yTAFII60 and yTAFII68, and also
       chromatin-remodeling complex          yTAFII25 and yTAFII90.
Mot1                                     STAGA (human)
       Repressor that binds TBP-             Human version of SAGA
       complex                           PCAF (human)
NC2                                          chromatin-remodeling complex with
       Global repressor that binds TBP       several histone-like TAFs
       (in absence of DNA)               TFTC
Nots                                         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 (yeast)
                                             chromatin-remodeling complex that
                                             contains the histone-like yTAFII17,
SAGA                                         yTAFII60 and yTAFII68, and also
       chromatin-remodeling complex          yTAFII25 and yTAFII90.
Mot1                                     STAGA (human)
       Repressor that binds TBP-             Human version of SAGA
       complex                           PCAF (human)
NC2                                          chromatin-remodeling complex with
       Global repressor that binds TBP       several histone-like TAFs
       (in absence of DNA)               TFTC
Nots                                         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
          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
    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
         Essential, highly conserved from yeast to humans
    Med-proteins
         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-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
    MBV4230

    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
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
       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
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
         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
    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
          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
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-størrelse
   implication: freely floating or anchored?

								
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