National Creative Research Initiative Center
for Genome Regulation, Samsung Biomedical Research Institute,
Sungkyunkwan University School of Medicine, Suwon
440-746,1 and Digital Genomics, Inc.,
Seoul 120-749,2 Korea, and
Laboratory of Molecular Cell Biology, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland
20892-42553
Received 26 October 2000/Returned for modification 6 December
2000/Accepted 5 January 2001
To decipher the mechanistic roles of Mediator proteins in
regulating developmental specific gene expression and compare them to
those of TATA-binding protein (TBP)-associated factors (TAFs), we
isolated and analyzed a multiprotein complex containing
Drosophila Mediator (dMediator) homologs. dMediator
interacts with several sequence-specific transcription factors and
basal transcription machinery and is critical for activated
transcription in response to diverse transcriptional activators. The
requirement for dMediator did not depend on a specific core promoter
organization. By contrast, TAFs are preferentially utilized by
promoters having a specific core element organization. Therefore,
Mediator proteins are suggested to act as a pivotal coactivator that
integrates promoter-specific activation signals to the basal
transcription machinery.
 |
INTRODUCTION |
Precise regulation of gene
expression is fundamentally required for a broad spectrum of
developmental processes in multicellular organisms. Although distinct
sequence-specific transcription factors are primarily responsible for
this regulation, transcriptional coactivator-corepressor proteins also
add a significant secondary layer to the regulation of gene expression.
A number of coactivator complexes have been identified in eukaryotes,
and their functions in gene activation at specific promoters have been
analyzed, primarily in vitro. However, the mechanism by which these
transcriptional coactivators regulate gene expression in living
organisms is not well understood.
Among eukaryotic transcriptional coactivators, two classes of proteins,
Mediator proteins and TATA-binding protein (TBP)-associated factors
(TAFs), are central to the process of transcriptional regulation. These
proteins were isolated as multiprotein complexes composed of more than
10 polypeptides and associate with the basal transcription machinery
(RNA polymerase II [Pol II] and TBP, respectively). Both complexes
interact with transcriptional activators and are required for
transcriptional activation in reconstituted in vitro systems (3,
8, 40). These facts suggest that Mediator and TAF complexes
function as coactivators by relaying transcriptional activation signals
from DNA-bound activators to the basal transcription machinery.
However, it has not been clearly determined whether Mediator and TAF
complexes contribute redundantly or distinctly to the transcriptional
activation process, nor have their mechanistic roles in Pol II
transcription been clearly deciphered.
Although both Mediator and TAF complexes were initially identified from
in vitro assays, recent genetic analyses in yeast suggest that TAFs do
not function as general coactivators under physiological conditions.
First, the depletion of various TFIID-specific TAFs does not have a
significant effect on transcriptional activation of most genes in yeast
(12, 28, 41). Second, yeast TAFII145/130 was
shown to function as a core promoter selectivity factor rather than a
general coactivator (36). These observations are in good agreement with earlier reports that certain TAFs, especially
Drosophila TAFII40 (dTAFII40),
dTAFII60, dTAFII150, and dTAFII250,
directly recognize core promoter elements (7, 39) and thus
are likely to act as promoter selectivity factors.
The Mediator complex was first identified in yeast as a multiprotein
complex (15, 16) containing functionally distinct modular
subassemblies composed of subunits with similar genetic properties
(19). Inactivation of individual Mediator proteins causes
widely variable effects on yeast gene expression, ranging from
deregulation of transcription of a small subset of genes to a
genomewide transcriptional defect (12). A number of
coactivator complexes related to yeast Mediator have been subsequently
isolated in mammals (5, 9, 13, 29, 31, 33, 37). Like yeast Mediator, mammalian complexes play a key role in regulating Pol II
transcription in vitro. However, their compositional and functional heterogeneity indicates that there has been a considerable increase in
the functional diversity of Mediator complexes during evolution.
In this study, we analyzed the function of the Drosophila
Mediator (dMediator) complex in order to gain insight into the
mechanism by which these coactivator complexes govern eukaryotic
transcriptional activation during Drosophila development. As
the first step toward this goal, we isolated a multiprotein complex
containing Drosophila homologs of yeast and mammalian
Mediator proteins. Our data shows that dMediator interacts physically
with several sequence-specific transcription factors and basal
transcription machinery and is critically required for transcriptional
activation in response to diverse transcriptional activators in vitro.
By contrast, the TAF complex is dispensable for transcription of
TATA-containing promoters in the presence of dMediator but is required
for transcription of TATA-less promoters that depend on an initiator
element (Inr) and downstream promoter element (DPE). Our results
suggest that dMediator functions as an essential coactivator complex
that integrates diverse gene-specific regulatory signals at specific
promoters, while TAF complex has its distinct role as a promoter
selectivity factor required for the expression of genes with a specific
type of core promoter.
| |
MATERIALS AND METHODS |
Plasmid constructs.
The transcription templates pG5E4T and
pE4T(dl-E)5 were provided by Michael Carey and Albert Courey,
respectively. The pG5-Adh40/10-G380, pNP3-Adh40/10-G380, and pAdh-G280
plasmids were provided by James Manley. pG5en and pG5AntpP2 were
constructed by cloning PCR-amplified sequences encoding nucleotides -45 to +155 of the engrailed (en) promoter and -45 to
+159 of the Antennapedia P2 promoter (AntpP2), respectively, into BamHI/EcoRI-digested pG5E4T.
To construct pGEX-Eve, the BamHI (blunted)-EcoRI
fragment from pET-GAL4-EveBCDEF-H6 (24), which encodes
amino acids 61 to 246 of Even-skipped, was inserted into
EcoRI/SmaI-digested pGEX-4T1 (Amersham Pharmacia Biotech).
Antibodies.
Antisera against dMediator homologs were
generated by immunization of rats with glutathione
S-transferase (GST) fusion proteins containing either
full-length (dMED6, dSOH1, dp34, Trfp, dp28b, and dSRB7) or partial
(amino acids 221 to 368 of dRGR1 and amino acids 1 to 168 of dTRAP80)
polypeptide. Polyclonal antibodies (Abs) used in immunoblotting and
immunoprecipitation were affinity purified from the antisera by antigen
affinity chromatography (10).
Buffers.
All buffers used in nuclear protein extraction,
column chromatography and dialysis contained 1 mM dithiothreitol (DTT)
and protease inhibitors (10 µM phenylmethylsulfonyl fluoride, 20 nM pepstatin A, 6 nM leupeptin, and 20 µM bisbenzamidine) unless otherwise specified. 2× lysis buffer contained 100 mM HEPES-KOH (pH
7.6), 500 mM potassium acetate, 2 mM EDTA, and 20% glycerol. Buffer
An contained 50 mM HEPES-KOH (pH 7.6), 1 mM EDTA, 10%
glycerol, and n mM potassium acetate. Buffer Sn
contained 20 mM HEPES-KOH (pH 7.6), 0.1 mM EDTA, 10% glycerol, and
n mM potassium acetate. Buffer Hn contains 100 mM
potassium acetate, 10% glycerol, and n mM potassium
phosphate (pH 7.6). Buffer IPn is identical to buffer
Sn except that it contains nonionic detergent IGEPAL CA-630 (Sigma) up to 0.2% and lacks DTT and protease inhibitors.
Purification of dMediator.
All operations were carried out
at 4°C. The amounts of dMediator proteins in nuclear extracts and
column fractions were monitored by immunoblotting with anti-dSOH1,
anti-dMED6, and anti-dSRB7 Abs. Drosophila embryo nuclei
were isolated as described previously (14) and suspended
in 2× lysis buffer (1 ml per g of embryos). The resulting suspension
was stirred with a magnetic bar for 30 min and centrifuged in an SW41Ti
rotor (Beckman) at 30,000 rpm. The clear interface solution was diluted
with buffer A0 to adjust the potassium acetate concentration to 100 mM
and then applied on a heparin-Sepharose column (1 ml per 20 mg of
protein) equilibrated with buffer A100. The resin was washed with
buffer A100, and bound proteins were eluted with buffer A300. The
eluted proteins were suspended in buffer S60 by dialysis and dilution
with S0 and applied on an SP-Sepharose column (1 ml per 20 mg of
protein) preequilibrated with buffer S60. The resin was washed with
buffer S60, and bound proteins were eluted with buffer S300. The eluted
proteins were suspended in buffer H10 by dialysis and applied on a
hydroxyapatite HTP column (1 ml per 2 mg of proteins) preequilibrated
with buffer H10. The resin was washed with buffer H10, and bound
proteins were eluted with a linear gradient from buffer H10 to buffer
H500. More than 80% of dMediator proteins were eluted at a potassium phosphate concentration between 350 and 400 mM even though there existed multiple minor peaks of dMediator proteins eluted at lower concentations. The major peak fractions of dMediator proteins (at 350 to 400 mM potassium phosphate concentrations) were pooled, dialyzed in
buffer S100, and applied on a Mono S column (1 ml) equilibrated with
buffer S100. The resin was washed with buffer S100, and bound proteins
were eluted with a linear gradient from buffer S100 to buffer S1000.
The peak fractions of dMediator proteins were pooled and incubated with
an anti-dSOH1 beads (0.1 ml) that were prepared as described elsewhere
(19). The beads were washed with buffer IP300, and bound
proteins were eluted twice with 0.1 ml of buffer IP100 containing 5 M
urea or with 0.1 ml of 0.1 M glycine-HCl (pH 2.5). The urea-eluted
dMediator fractions were dialyzed in buffer A100 and stored at
80°C.
In vitro transcription analyses.
Soluble nuclear fraction
was prepared from 0- to 12-h Drosophila embryos as described
elsewhere (14) except that 100 mM potassium acetate and
12.5 mM magnesium acetate were used in the nuclear extraction buffer
instead of potassium glutamate and magnesium chloride. For depletion of
endogenous dMediator and TFIID, 0.4 ml of soluble nuclear fraction was
incubated for 2 h at 4°C with 0.1 ml of protein G-agarose beads
retaining 0.1 mg of anti-dSOH1 or anti-dTAFII250 Ab. The
supernatants were transferred to a fresh tube containing an equal
amount of the Ab-bound beads and incubated for another 2 h. The
final supernatants were kept frozen at 80°C and used in
transcription assays. TFIID was prepared from Drosophila embryo nuclear extract using heparin-Sepharose, Q-Sepharose,
hydroxyapatite, and Mono S chromatographies, with dTAFII250
and TBP being monitored by immunoblotting. Transcription reactions were
performed in 25 µl containing 50 mM HEPES-KOH (pH 7.6), 50 mM
potassium acetate, 10 mM magnesium acetate, 5% glycerol, 0.2 mM EDTA,
1 mM DTT, 1 mM each nucleoside triphosphate, 20 U of RNasin (Promega),
40 µg of soluble nuclear fraction proteins, and 50 ng of plasmid template. After incubation for 30 min at 25°C, the reaction was terminated, and transcribed RNA was isolated and analyzed as described elsewhere (2). For G-less template assay, transcription
reactions were carried out as described above except that (i) 50 µM
UTP and 0.2 mM 3'-O-methyl-GTP (Pharmacia) were substituted
for 1 mM UTP and 1 mM GTP and (ii) 1 U of RNase T1 (Roche)
and 10 µCi of [
-32P]UTP were included. Transcribed
RNA was isolated as in primer extension assay and analyzed by
electrophoresis on a 6% polyacrylamide gel and autoradiography.
Protein-protein interaction assays.
GST fusion proteins used
in GST pulldown assays were expressed in Escherichia coli
DH5 and purified by glutathione-Sepharose chromatography (Amersham
Pharmacia Biotech) as described elsewhere (20). The GST
construct containing the Drosophila Pol II C-terminal domain
(CTD) was obtained from Arno Greenleaf. FLAG-Dorsal was expressed in
Sf9 cells from the baculovirus construct provided by Albert Courey and
purified as described elsewhere (43). GST and FLAG
pulldown assays were performed by incubating 40 µg of soluble nuclear
fraction proteins (Fig. 5B) or 0.2 µg of dMediator proteins in the
Mono S fraction 24 (Fig. 6A) and 10 µl beads retaining 1 µg of each
GST-fusion protein in 0.3 ml of IP300 buffer for 6 h at 4°C,
washing the beads with IP300 buffer, and eluting bound proteins with
sodium dodecyl sulfate (SDS) gel sample buffer. One-fifth of the input
proteins and the bound proteins were analyzed by immunoblotting.
The analyses of transcription factor binding to bead-immobilized
dMediator shown in Fig. 5C and 6C were carried out by incubating 35S-labeled proteins synthesized in TnT reticulocyte
lysates (Promega) with 10 µl of anti-dSOH1 beads retaining 0.1 µg
of dMediator. The beads were washed, and bound proteins along with the
input nuclear extracts were analyzed as in the GST pulldown assays.
| |
RESULTS |
Drosophila homologs of yeast and mammalian Mediator
subunits.
As a first step to identifying the dMediator complex, we
cloned the full-length Drosophila MED6 gene by PCR using
Drosophila melanogaster embryonic cDNA templates and
degenerate primers designed from the conserved regions of the yeast and
human MED6 proteins (Fig. 1A) (B. S. Gim and Y. J. Kim, unpublished data). Additionally, we searched
the Drosophila expressed sequence tag and genomic databases.
We identified the Drosophila homologs of five components with known yeast and mammalian counterparts (Fig. 1A) and of 11 Mediator components known only in mammals (Fig. 1B).
View larger version (94K):
[in this window]
[in a new window]
|
FIG. 1.
Amino acid sequence alignment of dMediator Homologs. (A)
Mediator homologs conserved in Drosophila, human, and yeast
(denoted by prefixes "d," "h," and "y," respectively). The
conserved residues are marked with boxes (black boxes for residues
where all three sequences are identical or similar; gray boxes where
two of the three are identical or similar). Only the conserved regions
are shown. (B) Metazoan-specific Mediator homologs. The conserved amino
acid sequences are marked as in panel A. hp28b and hp34 are the human
homologs of mouse Mediator proteins p28b and p34 (13).
|
|
To examine whether the dMediator homologs associate to form a complex
as do their yeast and mammalian counterparts, we immunoprecipitated Drosophila embryo nuclear extracts with affinity-purified
anti-dMED6 Ab. Immunoblotting revealed that dMED6 protein was
precipitated from the extract, together with dSRB7 and dSOH1 (Fig.
2A). When the extract was
immunoprecipitated with anti-dSOH1, the same three proteins were
coprecipitated (data not shown). This result suggests that these three
dMediator homologs associate to form a putative Mediator complex.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 2.
Identification of the dMediator complex. (A)
Immunoprecipitation of nuclear extracts with anti-dMED6 Ab. Equivalent
amounts of nuclear extract input, supernatant, and pellet of the
immunoprecipitation were resolved by SDS-PAGE and immunoblotted with
the antibodies indicated at the left. (B) Immunodepletion of nuclear
extracts with anti- -galactosidase (Mock) and anti-dSOH1 (-dSOH1).
The supernatants obtained after incubation were analyzed as in panel A. (C) Transcriptional activation of the E4 and Adh promoter constructs by
Gal4-VP16 in nuclear extracts. Before the in vitro transcription assay,
the nuclear extracts were immunodepleted with anti--galactosidase
(Mock) or anti-dSOH1 (-dSOH1). The amount (nanograms) of recombinant
Gal4-VP16 added to the reactions is indicated at the top. The
transcripts from the E4 templates containing five copies of the Gal4
DNA binding sites (G5-E4) and the alcohol dehydrogenase proximal (Adh)
templates with or without five copies of the Gal4 binding sites (G5-Adh
and Adh, respectively) are indicated by arrows at the left.
|
|
To examine whether this putative dMediator complex is the functional
analog of yeast and mammalian Mediator complexes, we examined whether
dMediator is required for transcriptional activation. To this end, we
produced dMediator-deficient nuclear extract by incubating
Drosophila embryo soluble nuclear extract with anti-dSOH1 Ab
or a control Ab specific for bacterial -galactosidase. Immunoblot analysis shows that the dSOH1, dMED6, and dSRB7 proteins in the nuclear
extract were efficiently depleted by anti-dSOH1 but not by the control
Ab (Fig. 2B). The immunodepletion of the putative Mediator complex from
the nuclear extract significantly reduced transcriptional activation of
Ga14-VP16 without affecting basal transcription, whereas no obvious
transcriptional defect was observed in the mock-depleted nuclear
extract (Fig. 2C). These results indicate that the dMediator complex is
both a structural and functional analog of the yeast and mammalian
Mediator complexes.
Purification of dMediator as a multiprotein complex related to
human Mediator complexes.
The diversity of Mediator-related
coactivator complexes identified in mammals demonstrates that these
complexes have great compositional and functional heterogeneity. To
test whether the dMediator homologs are part of a multiprotein complex,
Drosophila embryo nuclear extract was fractionated using
heparin-Sepharose, SP-Sepharose, hydroxyapatite, and Mono S
chromatographies (Fig. 3A). dMED6, dSOH1
and dSRB7 were cofractionated during all steps including the last Mono
S step (Fig. 3B). The ability of the Mono S fractions to restore
transcriptional activation to dMediator-depleted nuclear extract
comigrates exactly with the Mediator homologs on the Mono S column
(Fig. 3B).
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 3.
Purification of dMediator. (A) Outline of dMediator
purification. The numbers indicate the concentrations (millimolar) of
potassium acetate (heparin-Sepharose, SP-Sepharose, and Mono S) or
potassium phosphate (hydroxyapatite). (B) Immunoblot and
transcriptional (Trxn) analyses of Mono S fractions. Equal amounts of
the input (I) samples were loaded on the Mono S column, and the eluted
fractions (numbers above the lanes) were resolved by SDS-PAGE and
immunoblotted with the Abs indicated at the left. Mono S fractions 18 to 32 were added to the transcription reactions containing the
Gal4-VP16 protein and soluble nuclear fraction immunodepleted with
anti-dSOH1. (C) Transcription assay of dMediator activity. The
Gal4-VP16 protein and soluble nuclear fraction (SNF) immunodepleted
with anti--galactosidase (SNFmock) or anti-dSOH1
(SNF dSOH1) were used in transcription reactions as in
Fig. 2C. The Mono S peak fraction (#24) and immunopurified dMediator
(IP pellet) were added to the reactions as described above.
|
|
The dMediator complex was immunoprecipitated with anti-dSOH1 Ab from
the Mono S fractions. The immunopurified complex as well as the Mono S
peak fraction restored transcriptional activation to dMediator-depleted
nuclear extract (Fig. 3C, lanes 6 and 8). The restored transcription
levels (lanes 6 and 8) were lower than that of the mock-depleted
extracts (lane 2). This is presumably due to the partial loss of
dMediator activity caused by inactivation of one or more component(s)
in a subpopulation of dMediator during purification or, alternatively,
by concomitant depletion of other positive factors by the Ab treatment.
SDS-polyacrylamide gel electrophoresis (PAGE) and silver staining of
the immunoprecipitated dMediator revealed approximately 25 polypeptides
of molecular masses ranging from 14 to 240 kDa (Fig.
4A). The coprecipitated polypeptides were
identified by immunoblotting with Abs raised against several putative
dMediator homologs found from the Drosophila database searches (Fig. 1). These analyses confirmed the presence of dRGR1, dTRAP80, Cdk8, dMED6, dSOH1, dp34, Trfp (13), dp28b, and
dSRB7 in the dMediator complex (Fig. 4B, lane 3). When anti-Trfp Ab was
used for immunoprecipitation, these polypeptides were also coprecipitated (Fig. 4B, lane 4). Furthermore, mass spectrometric analyses confirmed the presence of dTRAP80, dMED6, and Trfp in the
dMediator complex (data not shown). Although we confirmed the identity
of only nine dMediator components, the Drosophila genome
database contained additional open reading frames homologous to other
yeast and mammalian Mediator proteins (Fig. 1). The unidentified components of the dMediator complex may be encoded by some of these
homologs. Given the presence of Cdk8 in the dMediator,
Drosophila cyclin C (23), the cyclin partner of
Cdk8 (18), is also likely a component of the dMediator
complex. We are currently raising Abs against these putative dMediator
proteins to confirm their presence in the dMediator complex.
Female-sterile-homeotic (Fsh), which is a Drosophila homolog
of the mouse Mediator component p96a (13), did not
cofractionate with, nor was it coprecipitated with, the dMediator
proteins (data not shown). This result suggests that Fsh does not
stably associate with the Mediator complex in Drosophila.
Immunoblot analysis also showed that no detectable amounts of
Drosophila CREB binding protein and Pol II were present in
the dMediator complex (data not shown).
View larger version (54K):
[in this window]
[in a new window]
|
FIG. 4.
Polypeptide composition and developmental expression
pattern of dMediator. (A) Immunoprecipitation of dMediator with
anti-dSOH1 beads. Proteins immunoprecipitated from buffer IP100 (lane
1) or the Mono S dMediator peak fraction (lane 2) were electrophoresed
on SDS-11.5% polyacrylamide gels and silver stained. Identified
dMediator components are indicated at the right. Immunoglobulin heavy
(IgH) and light (IgL) chains are indicated at the left. (B) Immunoblot
analysis of dMediator proteins immunoprecipitated with nonspecific
anti--galactosidase (NS), anti-dSOH1 (S), and anti-Trfp (T). The
input Mono S fraction (I) is shown for comparison. (C) Superose-6
chromatography of the dMediator complex. The Mono S peak fractions (I)
were fractionated on a Superose-6 column, and the fractions (numbers
above the lanes) were subjected to SDS-PAGE and analyzed by
immunoblotting with the Abs indicated at the left. The elution
positions of size markers (in kilodaltons) are indicated by arrows. The
size markers used were blue dextran (2,000 kDa; void volume),
thyroglobulin (667 kDa), ferritin (440 kDa), and bovine serum albumin
(66 kDa). (D) Developmental expression pattern of dMediator homologs.
Poly(A) RNA (4 µg per lane) from Drosophila embryos (E),
first (L 1)-, second (L 2)-, and third (L 3)-instar larvae, white
prepupae (PP), day 1 (P D1), day 2 (P D2), and day 3 (P D3) pupae, and
adults (A) were electrophoresed on a formaldehyde agarose gel and
probed with radiolabled DNA fragments derived from the dMediator genes
indicated at the left. rp49 was used as a positive control.
|
|
To estimate the apparent size of the dMediator complex in the Mono S
peak fraction, this eluate was further analyzed by Superose-6 gel
filtration chromatography. The dMediator complex that we identified migrated at a molecular size of 2 MDa (Fig. 4C). This size is comparable to that observed in the Superose-6 chromatographic analysis
of TRAP (9) and human Mediator (5). Taken
together, these results suggest that the dMediator complex we purified
is a true counterpart of the mammalian Mediator complexes.
Northern blot analysis of developmentally staged Drosophila
embryos, larvae, pupae, and adults for the expression of the dMediator genes revealed that many of the components had similar expression patterns (Fig. 4D). Large amounts of the dMediator transcripts were
maternally deposited to the embryos (Fig. 4D, lane 1). The expression
level gradually decreased during the larval stages (Fig. 4D, lanes 1 to
6) and then increased during mid-pupal metamorphosis (Fig. 4D, lane 7).
This result shows that the abundance of dMediator transcripts is
correlated with developmental activities.
Interaction of dMediator with gene-specific transcription
factors.
Previous studies in yeast and human cells have suggested
that transcriptional activator proteins interact with Mediator
complexes (5, 9, 11, 20, 29, 31). The requirement of
dMediator for the activated transcription in response to Gal4-VP16
(Fig. 2C) indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as
TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been
suggested to interact directly with transcriptional activators, we
examined the relative binding affinities of these coactivator complexes
with the VP16 protein. After incubation of nuclear extracts with an
excess of GST fusion protein beads containing either wild-type or
mutant (
456FP442) VP16 activation domain, the supernatants were
analyzed by immunoblotting with Abs against the components of the
coactivator complexes. Almost all of the dMediator proteins in the
nuclear extract (dTRAP80, dMED6, and Trfp) were removed by incubating
with GST-VP16 but not with GST-VP16
456FP442 (Fig.
5A, lanes 1 and 2). However, the amounts
of dGCN5, dTAFII40, dTAFII250, and dTBP in the
extract were not reduced at all by the incubation (Fig. 5A, lanes 1 and
2). When the proteins bound to the beads were analyzed, a large amount
of dMediator was retained only in the GST beads containing the
functional VP16 activation domain (Fig. 5A, lanes 4 and 5). The TFIID
and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the
amounts were less than 2% of the total amounts present in the extract
(Fig. 5A, lanes 4 and 5, and data not shown). These data indicate that, among known transcriptional coactivator complexes, Mediator is most
strongly bound to and most readily recruited to the activation domain.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 5.
Interaction of dMediator with sequence-specific
transcription factors. (A) Physical interaction of
Drosophila coactivator proteins with the VP16 activation
domain. Nuclear extract was incubated with GST beads containing the
wild-type (W) and 456FP442 (M) VP16 activation domains. Proteins in
unbound fractions (lanes 1 and 2), bead-bound proteins (lanes 4 and 5),
and the input extract (I; lane 3) were analyzed by SDS-PAGE and then
immunoblotted with the Abs specific for the coactivator proteins
indicated on the left. (B) Physical interaction of dMediator with
Dorsal and dHSF. FLAG-Dorsal and GST-dHSF fusion proteins were
immobilized on beads and used in pulldown assays as in panel. A. Binding of dMediator to each bead was monitored by immunoblotting with
anti-Trfp (lanes 1 to 3) and anti-dTRAP80 (lanes 4 to 6) Abs. (C)
Physical interaction of dMediator with several sequence-specific
transcription factors. dMediator was immobilized on anti-dSOH1 beads
and incubated with the 35S-labeled transcription factors
indicated at the left. The input sample (I) and proteins bound to blank
resin (R) and dMediator-immobilized beads (M) were separated by
SDS-PAGE and analyzed by autoradiography. (D) Requirement of dMediator
for transcriptional regulation by Dorsal, dHSF, and Even-skipped.
Plasmid templates containing the respective binding sites for Dorsal,
Gal4-dHSF, and Even-skipped were used in transcription reactions.
Transcriptional activation by Dorsal and Ga14-dHSF and transcriptional
repression by Even-skipped in soluble nuclear fraction (SNF)
immunodepleted with anti--galactosidase (Mock) and anti-dSOH1
(-dSOH1) were analyzed as in Fig. 2C. (dl-twi)5, five copies of the
Dorsal-Twist binding sites.
|
|
In addition to the model VP16 activator derived from herpesvirus,
dMediator interacts with Drosophila transcriptional
activators Dorsal and heat shock factor (dHSF). When dMediator complex
was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads
even after extensive washing (Fig. 5B, lanes 3 and 6). To extend this
study to other sequence-specific transcription factors important for
Drosophila development, we immobilized dMediator on protein
G-agarose beads through anti-dSOH1 Ab and examined the binding of
diverse 35S-labeled Drosophila transcription
factors. Bicoid, Krüppel, and Fushi-tarazu were retained
specifically on the dMediator beads, while Twist and Hunchback were not
(Fig. 5C). Therefore, dMediator functions as a binding target for many,
but not all, developmental specific transcription factors.
To evaluate the requirement of dMediator for activated transcription in
response to the Drosophila activator proteins that interact
with dMediator, we tested the ability of dMediator-deficient nuclear
extracts to support transcriptional activation by the Dorsal and
Gal4-dHSF proteins. The addition of Dorsal or Gal4-dHSF to
mock-depleted extract caused 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level
of transcriptional activation was reduced significantly (five- and
threefold activations, respectively) in nuclear extract that had been
depleted by anti-dSOH1 Ab (Fig. 5D, lanes 4 and 8). Therefore,
dMediator is absolutely required for transcriptional activation by all
the activators tested. Addition of purified dMediator back to depleted
extracts partially recovered activation by Dorsal and Gal4-dHSF (data
not shown) in much the same way as it did in the case of Gal4-VP16
(Fig. 3C).
One of the functions suggested for Mediator complexes is the negative
regulation of Pol II transcription (9, 37). This function
may have a mechanistic relationship with transcriptional repression by
sequence-specific transcription factors. Therefore, we examined both
physical and functional interactions between dMediator and the
Drosophila transcriptional repressor Even-skipped. Unlike
the strong interaction between dMediator and several transcriptional activators, dMediator did not bind Even-skipped protein (Fig. 5C). As
shown previously (24), the addition of Even-skipped protein to nuclear extract caused a 12-fold reduction in transcription from the template containing three copies of the Even-skipped binding
site upstream of the alcohol dehydrogenase proximal (Adh) promoter
(Fig. 5D, lane 10). Transcriptional repression by Even-skipped still
occurred in the dMediator-deficient nuclear extracts (lane 12).
Therefore, dMediator is not required for transcriptional repression by
the sequence-specific transcription factor Even-skipped.
Physical and functional interaction of dMediator with the basal
transcription machinery.
Although the yeast Mediator complex was
purified as part of Pol II holoenzyme complexes (15, 16),
the dMediator that we isolated does not contain Pol II or other general
transcription factors. However, we observed that a small fraction of
the dMediator comigrated with Pol II during several chromatographic
steps and also coimmunoprecipitated with Pol II from crude fractions at low salt concentrations (lower than 300 mM potassium acetate [data not
shown]). Therefore, we tried to confirm the interaction between dMediator and Pol II, in particular, the CTD of Pol II, by GST-pulldown assays. Purified dMediator was incubated with GST-CTD beads. Bound proteins were analyzed by immunoblotting with dMediator Abs. Like yeast
Mediator, dMediator bound specifically with GST-CTD (Fig. 6A).
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 6.
Interaction of dMediator with the basal transcription
machinery. (A) Interaction of dMediator with the Pol II CTD.
GST-pulldown assays were carried out as for Fig. 5A and B. dMediator
proteins in the input (Mono S fraction 24), GST-bound, and
GST-CTD-bound fractions were analyzed by immunoblotting with the
antibodies indicated at the left. (B) Phosphorylation of the Pol II CTD
by dMediator and TFIIH. GST fusion protein containing
Drosophila Pol II CTD was incubated with TFIIH (purified
from Drosophila embryo extracts and provided by Gaku
Mizuguchi) and/or dMediator and analyzed by immunoblotting with
monoclonal Abs that specifically recognize phosphorylated serine 2 (H5)
and serine 5 (H14) within the CTD. (C) Physical interaction of
dMediator with general transcription factors. 35S-labeled
general transcription factors, namely, TBP (T), TFIIB (B), TFIIE-L
(EL) and -S (ES), TFIIF-L (FL) and
-S (FS), TFIIA-L (AL) and -S (AS),
and TFIIS (S), were synthesized in reticulocyte lysates (left) and
incubated with bead-bound dMediator as for Fig. 5C. The sizes of
molecular weight markers are indicated at the left. Proteins bound to
dMediator beads were analyzed by SDS-PAGE and autoradiography
(right).
|
|
Because the Mediator-CTD interaction in yeast causes a stimulation of
CTD phosphorylation by TFIIH (15), we examined the effect
of dMediator on the phosphorylation of Drosophila CTD. The
phosphorylation states were monitored by immunoblot analyses with Abs
that recognize the phosphoserine residues at the second and fifth
serines of the CTD. Purified Drosophila TFIIH specifically phosphorylated the fifth serine residue of GST-CTD (Fig. 6B, lanes 2 and 3). On the other hand, dMediator phosphorylated both the second and
fifth serine residues (lanes 4 and 5). This may, at least in part,
result from the catalytic activity of Cdk8 present in the dMediator
complex. When both dMediator and Drosophila TFIIH were
present in the reaction, the level of the CTD phosphorylation at serine
5 is increased synergistically, whereas the phosphorylation at serine 2 by dMediator remained unchanged in the presence of TFIIH (lanes 6).
Increasing the amount of dMediator further stimulated the synergistic
serine 5 phosphorylation while that of TFIIH did not (lanes 7 and 8),
indicating that dMediator is the limiting factor for the synergistic
serine 5 phosphorylation in our assay condition. All of these data
suggest that although we could not observe a stable physical
interaction in vitro between dMediator and TFIIH (data not shown), they
interact functionally to stimulate the phosphorylation of serine 5 of
the CTD.
We next investigated interactions between dMediator and the other
Drosophila general transcription factors by incubating
individual 35S-labeled TBP, TFIIB, TFIIE, TFIIF, TFIIA, and
TFIIS subunits with dMediator-immobilized beads. SDS-PAGE analysis of
the dMediator-bound proteins revealed that TBP, TFIIB, the TFIIE small
subunit, the TFIIF large subunit, and TFIIS interacted strongly with
dMediator, whereas TFIIA, the TFIIE large subunit, and the TFIIF small
subunit did not (Fig. 6C). dMediator interacted with heteromeric TFIIE and TFIIF complexes assembled from the large and small subunits (data
not shown), presumably through binding to the TFIIE small subunit and
TFIIF large subunit, respectively. Therefore, dMediator exhibits a
broad spectrum of binding capacities for a subset of general
transcription factors and Pol II as well as sequence-specific transcription factors and may influence the function of these binding partners.
Distinct function of Mediator proteins and TAFs.
The general
requirement of dMediator in transcriptional activation in vitro raises
the question about what the functional relationship is between
dMediator and TFIID in transcriptional activation. Although the VP16
activator binding assay shown in Fig. 5A clearly indicated that
dMediator is the major binding target of the VP16 activator protein in
nuclear extracts, whether TAFs provide a redundant, synergistic, or
distinct function in transcriptional activation in the presence of
dMediator is not known. Therefore, we compared the requirement of
dMediator in Pol II transcription to that of TAFs, using
immunodepletion studies similar to that carried out in the human system
(30). We immunodepleted nuclear extract with an Ab against
dMediator (anti-dSOH1), TFIID (anti-dTAFII250), or
-galactosidase (control). Although we observed physical interaction
of dMediator with general transcription factors and Pol II (Fig. 6),
immunodepletion with anti-dSOH1 did not remove basal transcription
factors, TAFs, or Pol II from the nuclear extract (Fig.
7A, lane 2). This shows that the absolute
majority of dMediator proteins in nuclear extracts are not engaged in
the assembly of the Pol II holoenzyme complex.
View larger version (56K):
[in this window]
[in a new window]
|
FIG. 7.
Differential requirement of mediator proteins and TAFs
for transcriptional activation. (A) Immunodepletion of dMediators and
TFIID from nuclear extract. Extract was immunodepleted with
anti--galactosidase (M), anti-dSOH1 (S), and
anti-dTAFII250 (T). Each extract was analyzed by
immunoblotting to examine the relative amounts of the proteins
indicated at the left. (B) Transcriptional activation of the E4, Adh,
en, and AntpP2 promoter constructs by Gal4-VP16
in dMediator-deficient extracts. Extracts were immunodepleted with
anti--galactosidase or anti-dSOH1. Transcription assays were
performed and the results analyzed as for Fig. 3C. (C) Different
effects of TAF depletion on the activated transcription of the E4, Adh,
en, and AntpP2 promoter constructs. The addition
of Gal4-VP16, recombinant dTBP, and partially purified TFIID (TFIID
fr.) to each reaction is indicated by + on the corresponding
lanes.
|
|
The activated transcription by Gal4-VP16 from E4, Adh,
engrailed (en), and AntpP2 promoters
that bear upstream Gal4 binding sites was completely abolished by
immunodepleting, and partly restored by adding back, the dMediator
proteins (Fig. 7B, lanes 4 and 6). In contrast, the level of basal
transcription from the Adh promoter was not affected by the depletion
of dMediator (lane 3). Therefore, dMediator is specifically required
for transcriptional activation from all four templates, regardless of
their differential core promoter architecture (see below).
Nuclear extracts preincubated with the anti-dTAFII250 Ab
did not contain a detectable level of the dTAFII250
protein. dTAFII250, the largest component of the TFIID
complex, is the core scaffold upon which the other TAFIIs
are assembled to form a stable complex with TBP (42).
Hence, it is highly likely that the dTAFII250-deficient extracts lack the TFIID complex and the key functions played by TFIID-specific TAFs. On the other hand, they still possessed a residual
amount of TBP and dTAFII40 (Fig. 7A, lane 4). The residual TBP probably originated from other TBP-containing complexes such as SL1
and TFIIIB. However, these TBP molecules are not likely to participate
in Pol II transcription in an interchangeable fashion as these nuclear
extracts did not support either basal or activated transcription from
any of the promoters that we tested (Fig. 7C, lanes 3 and 4). The
residual dTAFII40 (lane 4) might originate from non-TFIID
TAF complexes such as the GCN5 HAT complex, which had not been removed
from nuclear extract by the dTAFII250 Ab treatment (lane
4). Addition of recombinant TBP to TFIID-depleted nuclear extracts
restored both basal and VP16-activated transcription from the E4 and
Adh promoters (Fig. 7C, lanes 5 and 6), demonstrating that TAFs are
dispensable for these promoters in the presence of Mediator. The E4 and
Adh promoters contain a TATA element at about the 30 position.
However, TBP alone was unable to support activated transcription from
the en and AntpP2 promoters (Fig. 7C, lanes 5 and
6). The en and AntpP2 core promoters differ from the above two promoters in that they lack a canonical TATA element but
instead consist of an Inr and a DPE (6). Therefore, TAFs, or at least dTAFII250, appear to have functions that are
redundant in the activated transcription of TATA-containing genes under the condition we used but critical for transcriptional activation from
TATA-less, Inr- and DPE-containing promoters. Indeed, addition of
partially purified TFIID to TFIID-depleted nuclear extracts restored
activated transcription from the en and AntpP2
promoters (lane 8). These observations reveal two distinct roles for
Pol II transcription, each played separately by Mediator proteins and
TAFs: mediation of activator responses and core promoter selectivity.
| |
DISCUSSION |
Mediator proteins and TAFs are differently required for Pol II
transcription.
dMediator is generally required for transcriptional
activation from both TATA-containing and TATA-less promoters (Fig. 7B) through direct communication with transcriptional activators. The
function of dMediator seems to be exclusively related to
sequence-specific transcription factors placed at upstream enhancer
elements. However, the requirement of TAFs, or at least
dTAFII250, in activated transcription appears to be
redundant in the in vitro transcription system we used and affected by
such factors as the core promoter organization or nucleosomal structure
of transcriptional templates. Several TAF components in the TFIID
complex indeed have biochemical activities and structural motifs
adequate for the recognition of specialized settings of transcription
templates. For example, certain TAFs recognize the Inr and DPE
sequences located in many Drosophila core promoters and
increase the stability of TFIID-promoter interactions (6, 7,
39). In addition, TFIID contains dTAFII250, which has a HAT catalytic activity (27) and also possesses a
histone octamer-like module comprising the histone H2B-, H3-, and
H4-like TAFs (8). Although not experimentally
demonstrated, these TAFs may have some roles in the transcriptional
regulation of nucleosomal templates.
Diverse sequence-specific transcription factors interact with and
depend on dMediator for transcriptional activation.
The
sequence-specific transcription factors which interact physically with
dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and
Fushi-tarazu (Fig. 5). These factors contain different types of
activation domains (acidic and glutamine-rich domains). Most of these
transcription factors have been shown to activate transcription either
constitutively or inducibly. It is noteworthy that dHSF interacts with
and requires dMediator for transcriptional activation (Fig. 5B and D)
because previous reports have shown that transcriptional activation by
HSF in yeast does not require the function of the Mediator protein Srb4
(21, 26). However, the recent finding that activation by
HSF depends on another Mediator protein, Rgr1 (22),
suggests that some function of Mediator is required for HSF-mediated
transcriptional activation in yeast, as well. As Rgr1, but not Srb4, is
conserved between yeast and Drosophila (Fig. 1A),
transcriptional activation by HSF might utilize the conserved Rgr1
components of the Mediator complexes.
Although some human Mediator complexes appear to have a negative effect
on activated transcription (9, 37), dMediator did not
exhibit such an activity in an in vitro transcription system
reconstituted with Drosophila transcription factors (data not shown). In addition, Even-skipped, a well-known
Drosophila transcriptional repressor, did not interact with,
or depend for its transcriptional repression on, dMediator (Fig. 5B and
D). Previous reports show that the repression domain of Even-skipped directly targets TBP (1, 38). We also confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped failed to
interact with dMediator (Fig. 5B and data not shown). Although Krüppel has a well-characterized repressor function in
Drosophila development, it can also act as a transcriptional
activator under certain conditions (35). Therefore, it is
more plausible that the dMediator-Krüppel interaction we observed
is a part of the mechanism for transcriptional activation rather than
transcriptional repression. Taken together with the fact that dMediator
was dispensable for basal transcription, the lack of defect of the
dMediator-depleted nuclear extracts on transcriptional repression by
Even-skipped protein suggests that dMediator is required mainly for the
mediation of transcriptional activation signals to the basal
transcription machinery. Very recently, developmental roles of certain
dMediator proteins found in the Drosophila genome database
have begun to be also identified in genetic studies (4).
Genetic interactions between dMediator proteins and a homeotic
regulator Sex combs reduced shown in that study
(4) implicate dMediator proteins as a transcriptional
activator-specific target critical for Drosophila development.
The interactions between basal transcription machineries and
dMediator may regulate the transcription initiation process.
Like
yeast Mediator, dMediator bound with the CTD repeats of
Drosophila Pol II (Fig. 6A). This implies that though
dMediator was purified separately from Pol II, these two complexes
indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS.
Such interactions may be involved in the regulation of Pol II
preinitiation complex assembly. Related with this idea, it has been
reported that in yeast, recruitment of general transcription factors
such as TBP, TFIIB, and TFIIH to active promoters requires the function
of Mediator (17, 25, 32). Also, TFIIE interacts with the
Mediator protein Gal11 (34). Further analyses will be
required to clarify whether these interactions, observed both in yeast
and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of
preassembled Pol II holoenzyme.
dMediator contains the protein kinase component Cdk8, which can
phosphorylate serine residues in the CTD. This catalytic kinase subunit
seems responsible, at least in part, for the Pol II phosphorylation by
dMediator (Fig. 6B). In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II
repeats, suggesting the presence of a functional interaction between
these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH
has been correlated with transcriptional activation processes
(20), the synergy in the serine 5 phosphorylation by TFIIH
and dMediator may be intimately linked with the regulatory effects that
the Mediator complex exerts on Pol II transcription.
J.-G.K. is a National Institutes of Health Fogarty International Center
Visiting Fellow and is supported by the Division of Basic Sciences,
National Cancer Institute. This work was supported by the Creative
Research Initiatives Program from the Korean Ministry of Science and
Technology to Y.-J.K.
| 1.
|
Austin, R. J., and M. D. Biggin.
1995.
A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking.
Mol. Cell. Biol.
15:4683-4693[Abstract].
|
| 2.
|
Becker, P. B.,
S. K. Rabindran, and C. Wu.
1991.
Heat shock-regulated transcription in vitro from a reconstituted chromatin template.
Proc. Natl. Acad. Sci. USA
88:4109-4113[Abstract/Free Full Text].
|
| 3.
|
Björklund, S., and Y.-J. Kim.
1996.
Mediator of transcriptional regulation.
Trends Biochem. Sci.
21:335-337[CrossRef][Medline].
|
| 4.
|
Boube, M. C.,
Faucher,
L. Joulia,
D. L. Cribbs, and H.-M. Bourbon.
2000.
Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification.
Genes Dev.
14:2906-2917[Abstract/Free Full Text].
|
| 5.
|
Boyer, T. G.,
M. E. D. Martin,
E. Lees,
R. P. Ricciardi, and A. J. Berk.
1999.
Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein.
Nature
399:276-279[CrossRef][Medline].
|
| 6.
|
Burke, T. W., and J. T. Kadonaga.
1996.
Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters.
Genes Dev.
10:711-724[Abstract/Free Full Text].
|
| 7.
|
Burke, T. W., and J. T. Kadonaga.
1997.
The downstream promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila.
Genes Dev.
11:3020-3031[Abstract/Free Full Text].
|
| 8.
|
Burley, S. K., and R. G. Roeder.
1996.
Biochemistry and structural biology of transcription factor IID (TFIID).
Annu. Rev. Biochem.
65:769-799[CrossRef][Medline].
|
| 9.
|
Gu, W.,
S. Malik,
M. Ito,
C.-X. Yuan,
J. D. Fondell,
X. Zhang,
E. Martinez,
J. Qin, and R. G. Roeder.
1999.
A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcriptional regulation.
Mol. Cell
3:97-108[CrossRef][Medline].
|
| 10.
|
Harlow, E., and D. Lane.
1988.
Antibodies: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 11.
|
Hengartner, C. J.,
C. M. Thompson,
J. Zhang,
D. M. Chao,
S.-M. Liao,
A. J. Koleske,
S. Okamura, and R. A. Young.
1995.
Association of an activator with an RNA polymerase II holoenzyme.
Genes Dev.
9:897-910[Abstract/Free Full Text].
|
| 12.
|
Holstege, F. C. P.,
E. G. Jennings,
J. J. Wyrick,
T. I. Lee,
C. J. Hengartner,
M. R. Green,
T. R. Golub,
E. S. Lander, and R. A. Young.
1998.
Dissecting the regulatory circuitry of a eukaryotic genome.
Cell
95:717-728[CrossRef][Medline].
|
| 13.
|
Jiang, Y. W.,
P. Veschambre,
H. Erdjument-Bromage,
P. Tempst,
J. W. Conaway,
R. C. Conaway, and R. D. Kornberg.
1998.
Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways.
Proc. Natl. Acad. Sci. USA
95:8538-8543[Abstract/Free Full Text].
|
| 14.
|
Kamakaka, R. T.,
C. M. Tyree, and J. T. Kadonaga.
1991.
Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos.
Proc. Natl. Acad. Sci. USA
88:1024-1028[Abstract/Free Full Text].
|
| 15.
|
Kim, Y.-J.,
S. Björklund,
Y. Li,
M. H. Sayre, and R. D. Kornberg.
1994.
A multiprotein Mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II.
Cell
77:599-608[CrossRef][Medline].
|
| 16.
|
Koleske, A. J., and R. A. Young.
1994.
An RNA polymerase II holoenzyme responsive to activators.
Nature
368:466-469[CrossRef][Medline].
|
| 17.
|
Kuras, L., and K. Struhl.
1999.
Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme.
Nature
399:609-613[CrossRef][Medline].
|
| 18.
|
Leclerc, V.,
J. P. Tassan,
P. H. O'Farrell,
E. A. Nigg, and P. Leopold.
1996.
Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II.
Mol. Biol. Cell
7:505-513[Abstract].
|
| 19.
|
Lee, Y. C., and Y.-J. Kim.
1998.
Requirement of a functional interaction between Mediator components Med6 and Srb4 in RNA polymerase II transcription.
Mol. Cell. Biol.
18:5364-5370[Abstract/Free Full Text].
|
| 20.
|
Lee, Y. C.,
J. M. Park,
S. Min,
S. J. Han, and Y.-J. Kim.
1999.
An activator binding module of yeast RNA polymerase II holoenzyme.
Mol. Cell. Biol.
19:2967-2976[Abstract/Free Full Text].
|
| 21.
|
Lee, D.-K., and J. T. Lis.
1998.
Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo.
Nature
393:389-392[CrossRef][Medline].
|
| 22.
|
Lee, D.-K.,
S. Kim, and J. T. Lis.
1999.
Different upstream transcriptional activators have distinct coactivator requirements.
Genes Dev.
13:2934-2939[Abstract/Free Full Text].
|
| 23.
|
Leopold, P., and P. H. Farrell.
1991.
An evolutionarily conserved cyclin homolog from Drosophila rescues yeast deficient in G1 cyclins.
Cell
66:1207-1216[CrossRef][Medline].
|
| 24.
|
Li, C., and J. L. Manley.
1998.
Even-skipped represses transcription by binding TATA binding protein and blocking the TFIID-TATA box interaction.
Mol. Cell. Biol.
18:3771-3781[Abstract/Free Full Text].
|
| 25.
|
Li, X.-Y.,
A. Virbasius,
X. Zhu, and M. R. Green.
1999.
Enhancement of TBP binding by activators and general transcription factors.
Nature
399:605-609[CrossRef][Medline].
|
| 26.
|
McNeil, J. B.,
H. Agah, and D. Bentley.
1998.
Activated transcription independent of the RNA polymerase II holoenzyme in budding yeast.
Genes Dev.
12:2510-2521[Abstract/Free Full Text].
|
| 27.
|
Mizzen, C. A.,
X. J. Yang,
T. Kokubo,
J. E. Brownell,
A. J. Bannister,
H.-T. Owen,
J. Workman,
L. Wang,
S. L. Berger,
T. Kouzarides,
Y. Nakatani, and C. D. Allis.
1996.
The TAFII250 subunit of TFIID has histone acetyltransferase activity.
Cell
87:1261-1270[CrossRef][Medline].
|
| 28.
|
Moqtaderi, Z.,
Y. Bai,
D. Poon,
P. A. Weil, and K. Struhl.
1996.
TBP-associated factors are not generally required for transcriptional activation in yeast.
Nature
383:188-191[CrossRef][Medline].
|
| 29.
|
Näär, A. M.,
P. A. Beaurang,
S. Zhou,
S. Abraham,
W. Solomon, and R. Tjian.
1999.
Composite co-activator ARC mediates chromatin-directed transcriptional activation.
Nature
398:828-832[CrossRef][Medline].
|
| 30.
|
Oelgeschläger, T.,
Y. Tao,
Y. K. Kang, and R. G. Roeder.
1998.
Transcription activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs.
Mol. Cell
1:925-931[CrossRef][Medline].
|
| 31.
|
Rachez, C.,
B. D. Lemon,
Z. Suldan,
V. Bromleigh,
M. Gamble,
A. M. Näär,
H. Erdjument-Bromage,
P. Tempst, and L. P. Freedman.
1999.
Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex.
Nature
398:824-828[CrossRef][Medline].
|
| 32.
|
Ranish, J. A.,
N. Yudkovsky, and S. Hahn.
1999.
Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB.
Genes Dev.
13:49-63[Abstract/Free Full Text].
|
| 33.
|
Ryu, S.,
S. Zhou,
A. G. Ladurner, and R. Tjian.
1999.
The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1.
Nature
397:446-450[CrossRef][Medline].
|
| 34.
|
Sakurai, H.,
Y.-J. Kim,
T. Ohishi,
R. D. Kornberg, and T. Fukasawa.
1996.
The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.
Proc. Natl. Acad. Sci. USA
93:9488-9492[Abstract/Free Full Text].
|
| 35.
|
Sauer, F., and H. Jäckle.
1991.
Concentration-dependent transcriptional activation or repression by Krüppel from a single binding site.
Nature
353:563-566[CrossRef][Medline].
|
| 36.
|
Shen, W.-C., and M. R. Green.
1997.
Yeast TAFII145 functions as a core promoter selectivity factor, not a general coactivity factor.
Cell
90:615-624[CrossRef][Medline].
|
| 37.
|
Sun, X.,
Y. Zhang,
H. Cho,
P. Rickert,
E. Lees,
W. Lane, and D. Reinberg.
1998.
NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription.
Mol. Cell
2:213-222[CrossRef][Medline].
|
| 38.
|
Um, M.,
C. Li, and J. L. Manley.
1995.
The transcriptional repressor Even-skipped interacts directly with TATA-binding protein.
Mol. Cell. Biol.
15:5007-5016[Abstract].
|
| 39.
|
Verrijzer, C. P.,
J.-L. Chen,
K. Yokomori, and R. Tjian.
1995.
Binding of TAFs to core promoter elements directs promoter selectivity by RNA polymerase II.
Cell
81:1115-1125[CrossRef][Medline].
|
| 40.
|
Verrijzer, C. P., and R. Tjian.
1996.
TAFs mediate transcriptional activation and promoter selectivity.
Trends Biochem. Sci.
21:338-342[CrossRef][Medline].
|
| 41.
|
Walker, S. S.,
J. C. Reese,
L. M. Apone, and M. R. Green.
1996.
Transcription activation in cells lacking TAFIIs.
Nature
383:185-188[CrossRef][Medline].
|
| 42.
|
Weinzierl, R. O. J.,
B. D. Dynlacht, and R. Tjian.
1993.
Largest subunit of Drosophila transcription factor IID directs assembly of a complex containing TBP and a coactivator.
Nature
362:511-517[CrossRef][Medline].
|
| 43.
|
Zhou, J.,
J. Zwicker,
P. Szymanski,
M. Levine, and R. Tjian.
1998.
TAFII mutations disrupt Dorsal activation in the Drosophila embryo.
Proc. Natl. Acad. Sci. USA
95:13483-13488[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
