132
RNA polymerase II as a control
coactivator complexes
panel for multiple
Michael Hampsey* and Danny Reinberg*?
1999
marks the 30th anniversary
of the reported
discovery
of o factor and the bacterial
RNA polymerase
holoenzyme.
In
1994, an RNA polymerase
II complex was discovered
in
yeast - mammalian complexes
were subsequently
identified.
Recent developments
regarding the composition
and
function of RNA polymerase
II complexes
suggest, however,
that the concept of the holoenzyme,
as defined in bacteria,
might not be relevant to eukaryotes.
Addresses
WHoward Hughes Medical Institute and the *Department of
Biochemistry, Division of Nucleic Acids Enzymology,
University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, Piscataway,
000545635,
USA
*e-mail: [email protected]
fe-mail: [email protected]
Current Opinion in Genetics & Development
New Jersey
1999, 9:132-l
39
http://biomednet.com/elecref/0959437X00900132
0 Elsevier
Science
Ltd ISSN
0959-437X
Abbreviations
ARC
activator-recruited
cofactor
CRSP
cofactor required for Spl activation
carboxy-terminal
heptapeptide
repeat domain
CTD
DRIP
vitamin D receptor-interacting
protein
GTFs
general transcription
factors
negative regulator of activated transcription
NAT
preinitiation
complex
PIG
RNAP
RNA polymerase
SMCC
SRBlmediator
coactivator
complex
TATA-binding
protein
TBP
TRAP
thyroid hormone receptor-associated
protein
Introduction
Bacterial
RNA polymerase (RNAP) core enzyme (a&3’)
is able to catalyze RNA synthesis de novo but is unable to
initiate accurate transcription
in a promoter-dependent
manner. This led to an assay for promoter-specific
initiation and the subsequent
discovery of CJ factor [1,2]. (J
factor associates with core RNAP in the absence of DNA
to form a holoenzyme
complex (a,BB’o), which, in contrast to the core RNAP, is capable of accurate initiation at
promoter DNA. Although
070 is the principal CJ factor,
alternative
cr factors have been identified
that direct
RNAP to structurally
distinct promoters [3]. Certain activator proteins target 0, although other subunits of RNAP
have also been identified
as activator
targets [4,5].
Accordingly,
the distinction
between bacterial core- and
holo-RNAP
is the presence of the (3 subunit, which is
essential for accurate transcription
initiation.
Purified eukaryotic RNA polymerases, similar to bacterial
RNAP, are unable to initiate promoter-specific
transcription [6]. A search for factors that would enable RNA
polymerase II (RNAPII) to accurately initiate transcription
led to the discovery of a family of factors that collectively
confer RNAPII
promoter specificity.
These factors are
known as the general transcription
factors (GTFs) and
include the TKIA-binding
protein (TBP), TFIIB, TFIIE,
TFIIF
and TFIIH
[7,8]. These factors are conserved in
structure and function among all eukaryotes [9]. Thus, the
GTFs are the functional counterparts of c~factors.
Do RNAPII and the GTFs constitute a holoenzyme that is
the eukaryotic equivalent of the bacterial RNAP holoenzyme? A subset
of the GTFs -TFIIB,
TFIIE,
TFIIF - has been found to interact with RNAPII directly and multiple interactions among the GTFs have been
defined [7,9]. Although these findings suggest the existence of an RNAPII-GTF
complex, this complex has
never been found on its own. RNAPII complexes containing the entire set of GTFs or a subset of GTFs, together
with other polypeptides,
however, have been isolated from
yeast and mammalian
cells [lO,ll].
Whether
these
RNAPII complexes constitute the eukaryotic counterparts
to bacterial RNAP holoenzyme is discussed below.
Yeast RNAPII complexes
Evidence for the existence of a eukaryotic RNAPII complex arose from the characterization
of SRB proteins in
yeast. Mutations
in SRB genes were identified
as suppressors of growth defects associated with truncations
in
the carboxy-terminal
heptapeptide
repeat
domain
(CTD) at the carboxyl terminus of the largest subunit of
RNAPII
[la]. All nine of the genetically
identified
SRB
proteins exist in a complex with RNAPII,
yet this complex includes only a subset of the GTFs [13-151. When
supplemented
with TBP and TFIIE,
this complex
is
capable of both accurate initiation
and activated
transcription
in response
to Ga14-VP16.
Furthermore,
depletion of Srb4 revealed a general requirement
for this
RNAPII complex in vlvo [16].
Additional
evidence for an RNAPII
complex came from
characterization
of the requirements
for transcriptional
activation in vitro. Although
purified
RNAPII
and the
GTFs are sufficient for promoter-specific
initiation,
this
system fails to respond to activators. This led to the identification
of a complex
that mediates
transcriptional
activation, termed mediator [ 171. The mediator is devoid
of RNAPII
but associates with the CTD and includes a
subset of the SRB proteins, a novel set of proteins denoted MEDs, and several polypeptides
identified
previously
as effecters of both positive and negative transcription
[ 18-201. Importantly, the negative effecters of transcription
exist in a subcomplex that can be dissociated without loss
of mediator function in vitro [ 181.
RNA polymerase
II as a control panel for multiple coactivator
complexes
Hampsey
and Reinberg
133
Figure 1
ledi
!X
C
Current
Op~mon tn Genetics
& Development
RNAPII
Pafl
complex
Modular nature of the yeast SRB/mediator
and Pafl complexes.
The
SRB/mediator
complex is depicted at top and its different
subcomplexes
are represented
in different colors. The subunit
composition
of each subcomplex
is also indicated. The composition
of
each subcomplex,
as well as interactions
within and between
subcomplexes,
were inferred from the studies of Lee and Kim [79]. The
Srb8-Srbl
1 subcomplex
is derived from genetic studies and might
include other polypeptides
[11,22]. The functions of the SRBcontaining
subcomplexes
are mediated through the RNAPII CTD,
whereas the Pafl subcomplex
apparently interacts with RNAPII
independently
of the CTD. RNAPII is depicted in green and the CTD is
shown as a string of spheres.
The SRB and mediator complexes are structurally similar
but not identical. Notably absent from the mediator complex are Srb8-11
[LX)], which include
the SrblO/ll
kinase/cyclin
pair [Zl]. Interestingly,
these SRB subunits
were identified
in several genetic screens as negative
effecters of transcription [ZZ]. Therefore the structural differences between the SRB and mediator complexes can be
accounted for by the different purification
strategies used
to isolate the respective complexes. Whereas mediator was
purified on the basis of an assay for transcriptional
activation, the purification of the SRB complex was independent
of a functional
requirement.
This difference
can also
account for the presence of the SWI/SNF
chromatinremodeling
complex in the SRB complex [23] and its
absence from the mediator [ZO], since the functional assay
used to purify mediator is based on naked DNA templates.
Galll, and a novel subcomplex that includes Pafl, Cdc73,
Hprl and Ccr4 [24,25]. This Pafl complex was purified
using antibodies recognizing the CTD of RNAPII and it is
unknown whether this procedure displaced polypeptides
whose interaction with RNAPII is mediated through the
CTD such as SRB/mediator. Also, it has not been reported
whether expression of the genes regulated by this complex
is independent of the SRB/mediator.
Recent results demonstrate the existence of yet another
complex that interacts with RNAPII to regulate expression
of an apparently unique set of genes. This form of the
RNAPII complex is devoid of SRB and other mediator subunits but includes a subset of the GTFs (TFIIB, TFIIF),
Two recent reports [26”,27’]
demonstrate that transcription of a subset of genes - CliPI, HSPK? and SSA4 - can
occur in the absence of SRB/mediator.
Interestingly,
SRB/mediator-independent
transcription
was also independent of the TFIIH
kinase activity. These results are
consistent with the model suggesting that the SRB/mediator functions through the CTD and its activity is affected
by CTD phosphorylation
[28].
These results collectively
suggest the presence of modular
subcomplexes
that associate with RNAPII
to affect the
response to specific transcription
factors. The association
of such modules with RNAPII would be dictated by the
134
Chromosomes
and expression
mechanisms
Figure 2
NAT
Current
Op~nm
I” Genetics
& Developn
Modular nature of coactivator
ARC/DRIP complex includes
complexes
in mammalian
cells. The
the CRSP complex (blue), which
mediates
Spl activation
independently
of ARC/DRIP.
The subunit
composition
of the repressive
NAT complex is also shown. The
subunits common to ARC/DRIP
and NAT are depicted
by common
colors. Subunits
of both complexes
that are homologous
to yeast
SRBlmediator
components
are labeled. The ability of CBP/p300
to
interact with ARC/DRIP
is shown. The indicated
subunit
compositions
were defined for ARC L48.1, DRIP [43-l, CRSP 145’1
and NAT [34*].
physiological
needs of the cell and by the nature of the
genes to be transcribed. Accordingly,
multiple
RNAPII
complexes would exist that are capable of integrating both
positive and negative regulatory signals.
ments imposed during
used to define subunit
Mammalian
RNAPII complexes
The discovery of the yeast RNAPII
complex was followed immediately
by the identification
of RNAPII
complexes in mammalian cells [29,30]. Moreover, multiple complexes,
ranging in size from 1.5 to 4 Mda, were
identified
in independent
laboratories
(for reviews, see
[lo, 111). These complexes are heterogeneous
in composition, but all contain common subunits that include
homologs of yeast SRB/mediator
proteins. Furthermore,
each of these complexes includes different subsets of the
GTFs,
with one complex
containing
the entire set
including
TFIID
[31]. The heterogeneity
among these
complexes extends to different subsets of SRB/mediator
polypeptides,
as well as factors that affect chromatin
structure
and nucleic acid metabolism.
These differences are likely to reflect several different parameters,
including
the different
methodologies
used to isolate
such large complexes,
the different functional
require-
purification,
composition.
and even the criteria
Purification
of human RNAPII
complexes
by conventional
one
case
following
chromatography
- in
co-elution of polypeptides
with RNAPII without imposition of function, and in the other case by assaying for
transcription
from naked DNA templates - resulted in
the isolation of two distinct forms of human RNAPII [32].
The former complex
contained
chromatin-remodeling
activities,
including
SWI/SNF
and the histone acetyltransferases CBP and PCAF, but was devoid of G’I’Fs.
The other complex did not include factors that modify
chromatin but contained a subset of SRB/mediator
subunits and GTFs, along with other polypeptides
that
mediate transcriptional
activation
[30,32]. The coactivator complex was displaced from RNAPII
by antibodies
against the CTD [30]. Furthermore,
transcriptional
activation was independent
of the PC4 coactivator, which is
required
for Gal4-VP16mediated
activation
by core
RNAPII.
These properties are reminiscent
of the yeast
SRB/mediator
complex. Thus, the differential
functional
requirement
resulted in the purification
of two distinct
RNA polymerase
forms of mammalian RNAPII complexes,
ilar to that reported
for yeast.
II as a control panel for multiple coactivator
a scenario sim-
Whether the SWI/SNF complex is indeed associated with
RNAPII
has been the subject of controversy
[2X,23].
Nonetheless, SWI/SNF has been found in human RNAPII
complexes
following
different
purification
strategies
[32,33]. Co-purification
of RNAPII
with the human
homolog of the yeast SrblO/ll
complex (Cdk8/cyclin
C),
yielded a complex that included SWI/SNF subunits, the
histone acetyltransferase
coactivator CBP/p300 and other
polypeptides
[33]. Furthermore,
anti-Cdk8 affinity purification of fractions enriched for SRB/mediator
yielded two
complexes, one including SWI/SNF and the other devoid
of SWI/SNF but including polypeptides
that affect transcription activation [32,34’].
The Cdk8-containing
complex impaired
transcriptional
activation; accordingly, this complex is designated NAT,
for negative regulator of activated transcription
[34’]. The
subunit composition
of NAT includes homologs of the
yeast mediator complex, as well as Rgrl and SrblO/ll,
which were identified
as negative regulators of transcription [Zl]. ‘The SrblO/ll
heterodimer
[21,3.5] and the NAT
complex
[34’] phosphorylate
the CTD
of RNAPII.
However, in contrast to the TFIIH
kinase, which phosphorylates
the CTD
only after assembly
of the
preinitiation
complex [36], SrblO/ll
and NAT phosphorylate the CTD
prior to assembly of the preinitiation
complex (PIC) [34’,35]. As the PIC is assembled from the
non-phosphorylated
form of RNAPII, the temporal specificity of SrblO/ll
and NAT phosphorylation
might account
for their negative effects on transcription
[34’,35].
complexes
Hampsey
and Reinberg
135
limiting TFIIH
concentrations. It was suggested that the
negative effect of SMCC on activated transcription might be
mediated through phosphorylation
of the general coactivator PC4. In support of this conclusion, substitution of PC4
by the coactivator PC2 eliminated
the negative effect of
SMCC on transcription.
However, PC2 stimulated
basal
transcription without a clear effect on activation, thereby
questioning whether the negative effect of SMCC is indeed
mediated through PC4 [38]. Indeed, as the coactivator function of the yeast SRB/mediator
[40] and human RNAPII
complexes [30,31] are independent
of other cofactors, a
‘true’ mammalian mediator complex would be expected to
be independent of other cofactors. Nonetheless, the ability
of SMCC to function as a coactivator, albeit in the context of
limiting TFIIH
and in the presence of PC4, is intriguing
and underscores the interplay between coactivators and
GTFs, and the potential of RNAPII complexes to function
as both positive and negative effecters of transcription.
The SMCC complex includes all of the reported NAT subunits, including
three subunits of the TRAP complex:
TRAPlOO, TRAP170 and TRAP220 [38]. TRAP is a coactivator complex isolated on the basis of its interaction with
the thyroid hormone receptor in a ligand-dependent
manner [41]. TRAP170 is the mammalian homolog of Rgrl [38],
a negative effector of transcription contained in the yeast
SRB/mediator complex [ 181. Another coactivator complex,
DRIP-which
was isolated on the basis of its ability to
interact with the vitamin D, receptor in a l&and-dependent
manner-includes
novel subunits as well as subunits common to the NA’I’/SMCC
and TRAP complexes [42,43’].
Whereas the yeast SrblO/ll
heterodimer
targets Ser-5 of
the CTD [35], the human NAT complex phosphorylates
both Ser-2 and Ser-5 [34’]. Ser-2 has been genetically
linked to Srb9 [37], which f uric.t’ions in repression in vZv0
and is genetically
related to SrblO/ll
[Z?]. Although the
CTD is clearly a target of SrblO/ll
in both yeast and
human, it is not the sole target of the NAT complex, as
NAT associates physically and functionally
with RNAPII
lacking the CTD. Nonetheless,
the CTD regulates the
association of NAT with RNAPII. Phosphorylation
of the
CTD precludes the association with NAT [34’].
The theme that begins to emerge from the characterization
of these complexes is the presence of common subunits in
distinct complexes. Perhaps the specificity is mediated by
unique subunits that interact with distinct activators.
Consistent with this idea, one of the subunits present in the
TRAP and DRIP complexes. TRAP220/DRIP205,
interacts in a l&and-dependent-manner
with multiple nuclear
hormone receptors [43’,44]. Moreover, the search for a
coactivator for the glutamine-rich
Spl activator uncovered
yet another coactivator
complex - CRSP [45’]. CRSP
shares subunits with NAT/SMCC,
TRAP and DRIP, and
includes
a homolog
of the Med7 subunit
of yeast
SRB/mediator complex, as well as novel polypeptides.
A complex with strikingly similar structural and functional
properties to NAT has independently
been identified, using
affinity purification and cell lines that stably express FLAGtagged Srb7, SrblO or Srbll [38]. In contrast to NAT, this
complex,
designated
SMCC (SRB/mediator
coactivator
complex), can also mediate transcriptional
activation. A
mouse complex containing a subset of SRB and MED proteins
stimulates
that
TFIIH-dependent
CTD
phosphorylation
has also been described, although potential
effects on transcriptional
activation were not reported [39].
‘The activities of the SMCC complex appear to be dependent upon the specific coactivator used in the assay, or upon
The structural relationships
among these independent
coactivator complexes
suggest that specific coactivators
exist to respond to specific activators, yet these complexes
include common subunits. This leads to the prediction
that coactivators isolated on the basis of their requirement
to mediate simultaneously
the response to multiple activators will include previously defined coactivator complexes,
together with specific polypeptides
that can integrate each
activator function. A likely candidate for a polypeptide
with integrating
function is CBP/p300, which apparently
has the ability to physically and functionally
interact with
many activators and coactivators [46]. Moreover, CBP/p300
136
Chromosomes
and expression
mechanisms
also interacts with RNAPII, and this interaction is specific
for the initiation-competent
form of RNAPII [32]. A recent
report analyzing the synergistic effect of two activators,
Spl and SREBP, described a coactivator complex containing CBP [47]. Importantly,
the synergy between these
activators required chromatin, and the function of CBP in
this system was apparently
independent
of its histone
acetyltransferase
activity. This complex,
termed ARC,
includes all of the polypeptides
present in CRSP and
DRIP, in addition to novel polypeptides
[48’].
A question that arises from this analysis is whether these
coactivator complexes require the TAF subunits of TFIID.
Thus far, the effects of each of these coactivator complexes
in mammalian cells is dependent on the TFIID
complex.
This is in contrast to the yeast SRB/mediator complex, which
mediates transcriptional activation in a TBP-dependent
but
TAF-independent
manner. The difference between the
requirements for these two systems is unknown but might
reflect differences in genome complexity [49].
Functional
redundancy
A common theme to emerge from the studies summarized
here is that mediators exist as multiple complexes with
shared subunits. This raises the possibility of overlapping
functions.
Indeed,
genetic
studies have shown that
SRB/mediator, the SAGA histone acetyltransferase complex,
and the SWI/SNF complex have overlapping functions in
viva [50]. Whether these complexes are also functionally
redundant with TFIID has not been reported. In the yeast in
vitro system, however, TAFs are dispensable for activation,
apparently replaced by the SRB/mediator
complex. This
observation is also supported in viva, where certain TAFs are
not generally required for activation [51,52]. This raises the
possibility that coactivator function in mammalian systems
might be TAF-independent
under certain conditions. Two
recent reports have addressed this issue. In one case, activator-dependent transcription was observed in a reconstituted
system in the absence of TAFs [53,54]; however, this activity is dependent upon PC4 which, at high concentrations,
inhibits transcription.
Therefore,
the TAF-independent
transcription observed in those studies might reflect antirepression rather than true activation. In the other case,
robust activation was observed using a HeLa-derived
extract
that had been immuno-depleted
of TAFs [SS’]. Presumably,
the TAF requirement
for co-activation was replaced by a
redundant activity in the crude HeLa extract. Thus, there
appears to be functional redundancy among coactivator complexes in both yeast and mammalian systems.
Further evidence for functional redundancy among coactivator complexes is the presence of common TAF subunits
in SAGA and TFIID
[K-59].
Another TAF-containing
complex, but devoid of TBP, was shown to be functionally
redundant with TFIID in vitro [60]. Interestingly, while this
complex is devoid of TBP, its function at TATA-containing
promoters is dependent upon this element even though the
complex also functions at naturally occurring TATA-less
promoters.
These experiments,
together
with recent
descriptions of TBP-related
factors in unique complexes
[61], provide clear evidence for functional
redundancy
among TBP or TBP-related cofactor complexes.
Recruitment of transcription
to promoters
complexes
Formation of transcription complexes in bacteria is a relatively simple process involving promoter recognition by the
RNAP holoenzyme
complex. In some cases, activators
stimulate transcription by interacting directly with RNAP
subunits, primarily the DNA binding domains of the a or c~
subunits [S]. This latter scenario also applies in eukaryotic
systems, where transcription is stimulated by activator contact with GTFs [7], or with an RNAPII
subunit [62]. In
keeping with the more complex nature of eukaryotes, however, transcription
in yeast can involve recruitment
of at
least two distinct complexes - TFIID
and SRB/mediator.
One model for stimulation
of PIC assembly in yeast is
activator-mediated
recruitment
of TBP. Strong support
for this model came from tethering experiments
involving fusions of the DNA-binding
domain of either Gal4 or
LexA to TBP. These fusion proteins bypass the activator
requirement
for transcription
[63-651. When altered
forms of TBP were analyzed in the bypass experiment,
however, two classes of mutants were identified.
TBP
derivatives
that were defective
for promoter
binding
were rescued by tethering,
whereas
the activation
defects of other derivatives
could not be rescued [66].
These results led to the proposal that activation
by
recruitment
can be a two-step process, with one step
occurring subsequent to TBP recruitment
[67].
The two-step model for activation is supported by other
recruitment
experiments.
In these cases, tethering
of
either the Gall1 or SrbZ components of the SRB/mediator complex,
or TFIIB,
was sufficient
for activation
[68-701. As expected for a two-step recruitment
model,
activation
in these experiments
remained
dependent
upon a functional TATA box [70]. The physiological
relevance of these results is supported
by recent studies
demonstrating
interactions
between activators and Srb4
[71], Med6 [72] and Gall 1 [73]. Furthermore,
strong support for the validity
of the artificial
recruitment
experiments
is provided by fusion of activation domains
to components of either TFIID
or SRB/mediator,
which
in every case failed to stimulate transcription
[74].
Direct biochemical
support for this model comes from
template-immobilization
experiments
where PIC assembly occurred by at least two independent
steps, one
involving
TFIID
recruitment
independent
of RNAPII,
the other involving RNAPII recruitment
in an SRB- and
CTD-dependent
manner [75”]. A similar conclusion was
reached by analyzing enhanceosome-dependent
transcription in a mammalian system [76]. The yeast experiments
suggest the involvement
of at least one additional
step
RNA polymerase
II as a control
subsequent to TFIID
and RNAPII recruitment
for transcription. This implies that formation
of the PIC by
activator-mediated
recruitment
is not sufficient for transcription but that steps subsequent
to PIC formation are
also targets for activators.
panel
for multiple
References
In the year to come, the distinction between bacterial and
eukaryotic holoenzyme complexes will become even clearer. Microarray analysis of the entire yeast genome using
mutants defective in components of RNAPII holoenzyme
complexes will define the range of genes affected by specific subunits or subcomplexes.
Furthermore,
the use of
more physiological
transcription
assays, including
chromatin templates,
is likely to uncover new coactivator
complexes comprising both novel and previously defined
components. The characterization
of these complexes and
the genes affected by them is certain to provide insight
into the complex mechanisms of transcriptional
control of
gene expression.
Acknowledgements
We are grateful to L Freedman, R Roeder and R Tjian for communicating
results prior to publication. We also thank R Ebright, K Struhl and R Tjian
for valuable discussions, and R Ebright for wmments on the manuscript. D
Rcinberg is supported by grants from the National Institutes
of Health
(GM37120 and GM48518) and the Howard Hughes Medical Institute. M
Hampsey is supported by National Institutes of Health grant GM39484.
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and mammalian
systems but are these true holoenzyme
complexes? RNAP holoenzyme,
as defined in bacteria, is
capable of promoter-specific
recognition
and transcription initiation.
In eukaryotes,
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has been described for only a single
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includes
all components
necessary for transcription.
Clearly, different
complexes
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an
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transcribing
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