Transcript TAFs and the Mediator

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) 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, 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 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
 1.
main function: interaction with
activators


Physical contact found between TAFs and specific activators
TAF-activator contact: each type of activator contacts a particular
basal trx.app.
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
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 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
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


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
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 knockedout - 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-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 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-størrelse
implication: freely floating or anchored?