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Molecular and Cellular Biology, September 1998, p. 5355-5363, Vol. 18, No. 9
Howard Hughes Medical Institute, Division of
Nucleic Acid Enzymology, Department of Biochemistry, Robert Wood
Johnson Medical School, University of Medicine and Dentistry of New
Jersey, Piscataway, New Jersey 088541;
DNAX Research Institute of Molecular and Cellular Biology, Palo
Alto, California 943043; and
Laboratory of Molecular Growth Regulation, National
Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 208922
Received 13 May 1998/Accepted 16 June 1998
We have isolated a human RNA polymerase II complex that
contains chromatin structure remodeling activity and histone
acetyltransferase activity. This complex contains the Srb proteins, the
Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in
addition to RNA polymerase II. Notably, the general transcription
factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity
chromatography using antibodies directed against CDK8, the human
homolog of the yeast Srb10 protein. Protein interaction studies
demonstrate a direct interaction between RNA polymerase II and the
histone acetyltransferases p300 and PCAF. Importantly, p300 interacts
specifically with the nonphosphorylated, initiation-competent form of
RNA polymerase II. In contrast, PCAF interacts with the
elongation-competent, phosphorylated form of RNA polymerase II.
Transcription is a key step at which
gene expression is regulated. The first step of transcription involves
the recognition of promoter DNA sequences and the formation of a
transcription initiation complex (54). DNA in the cell is
associated with histone proteins to form nucleosomal arrays that are
compacted into a highly ordered protein-DNA structure known as
chromatin (32). Chromatin has been shown to be a barrier to
transcription, as well as to most processes requiring access of
proteins to DNA (30, 76). Increasing evidence suggests that
the accessibility of factors to DNA packed into chromatin is an
important event in the regulation of transcription. Activities that
increase the accessibility of proteins to DNA packaged into chromatin
have been characterized (70). The Swi-Snf complex
can remodel nucleosome structure to facilitate the access of
DNA-binding proteins to DNA (34, 58). Other
nucleosome-remodeling factors have also been isolated from yeast
(RSC [5]), Drosophila (NURF
[69], CHRAC [72], and ACF
[24]), and human (34, 74) cell
extracts. The conservation of these machineries emphasizes the
importance of their function.
Direct covalent modification of core histones has also been implicated
in the activation of transcription. An increase in histone
acetylation correlates with increased levels of transcription (18,
22). The finding that well-studied coactivators, such as Gcn5
(4), p300/CBP (3, 53), and TAFII250
(44), are histone acetyltransferases is consistent with a
role for histone acetylation in transcriptional activation. Conversely,
the finding that histone deacetylases are components of a Sin3
repressor complex (1, 21, 23, 26, 46, 80) also agrees
with the observation that histone acetylation plays a key
role in regulating transcription. However, whether histones are the
actual and only targets of these acetyltransferases and deacetylases
remains to be determined.
Histone acetyltransferases and deacetylases have been demonstrated to
interact with sequence-specific transcription factors (1, 13, 21,
23, 26, 46, 73, 80) and with components of the general
transcription machinery (25, 73). This suggests a model
whereby these activities can be recruited to promoters to alter the
acetylation state of promoter-bound histones. This, in turn, would
increase or decrease promoter accessibility (18).
RNA polymerase II (RNAPII) is a 12-subunit complex in which the largest
subunit contains a carboxy-terminal domain (CTD) composed of a
heptapeptide repeat sequence (YSPTSPS) ich in amino acids that can be phosphorylated (78).
The phosphorylation of the CTD is highly regulated and can modulate the
association of proteins with RNAPII (50, 65). The RNAPII
that contains an unphosphorylated CTD (IIA form) is able to form
transcription initiation complexes (10, 14, 39). The
elongating RNAPII contains a highly phosphorylated CTD (IIO form)
(14, 52, 57).
The CTD of RNAPII is essential for viability (2, 79). In
yeast, the cold-sensitive growth phenotype associated with partial truncation of the CTD led to the isolation of the SRB
(suppressor of RNA polymerase B) family of genes (51).
The Srb proteins are associated with RNAPII and a subset of general
transcription factors forming an RNAP holoenzyme complex
(31). A similar complex, referred to as the mediator,
differing slightly in polypeptide composition has also been isolated by
using a biochemical approach to search for activities that mediate the
response to transcriptional activators (29). This
complex not only mediates activated transcription but also
increases basal transcription and allows increased phosphorylation of
the CTD. Several independent studies have also suggested negative roles
for the Srb-mediator complex in transcription (7, 20, 33,
66).
Complexes similar to the yeast RNAPII holoenzyme have been isolated
from mammalian cells (9, 40, 56). However, the mammalian
complexes show greater heterogeneity than the ones derived from yeast.
The mammalian complexes contain a variety of different proteins in
addition to Srb homologs and general transcription factors (GTFs)
(9, 40, 56), such as proteins involved in recombination and
DNA double-strand break repair (40), transcription-coupled repair (71), and RNA processing (such as polyadenylation and splicing factors) (43); elongation factors (56);
coactivators mediating the response to Gal4-VP16 (40, 56),
Gal4-SP1 (56), and the human immunodeficiency virus type 1 transactivator Tat (12, 68); coactivators that contain
histone acetyltransferase activity such as CBP (47); the
breast cancer tumor suppressor gene product BRCA1 (62); and
RNA helicase A (RHA) (48). As it became clear that chromatin
remodeling is an important step in the regulation of transcription, the
existence of RNAPII complexes containing activities capable of
modifying chromatin was predicted. Indeed, a yeast RNAPII complex
associated with the Swi-Snf chromatin remodeling complex has been
reported (75). However, the existence of this complex has
been controversial and a similar complex has not been detected in
mammals.
Here we report the isolation of an RNAPII complex that contains human
Srb proteins and factors that modify chromatin structure, including the
Swi-Snf complex and the histone acetyltransferases CBP and PCAF.
Purification of RNAPII complexes.
RNAPII complexes were
purified as follows. HeLa cell nuclear extracts (2.3 g) were
fractionated on a 500-ml phosphocellulose (Sigma) column which was step
eluted with buffer C (20 mM Tris-HCl [pH 7.85], 0.2 mM EDTA, 10 mM
Western blot analysis.
Blots were incubated with 3%
(wt/vol) nonfat dry milk for 1 h at room temperature with shaking.
Following three washes with TTBS (10 mM Tris-HCl buffer [pH 7.5], 0.2 M NaCl, 0.05% [vol/vol] Tween 20), blots were incubated with primary
antibodies in 0.1% (wt/vol) bovine serum albumin containing TTBS for
2 h at room temperature with gentle agitation. Blots were washed
again with TTBS and incubated with secondary antibodies conjugated to
alkaline phosphatase (Promega) or horseradish peroxidase (Bio-Rad) for 30 min at room temperature. Blots were washed with TTBS and developed according to the manufacturer's protocol (Bio-Rad or Boehringer Mannheim).
Immunoprecipitation.
Affinity-purified antibodies
(approximately 2 µg) were incubated with 15 µl of protein A-agarose
beads (Repligen) for 30 min at room temperature. After a washing with
buffer E (20 mM Tris-HCl [pH 7.85], 0.2 mM EDTA, 1 mM dithiothreitol
[DTT], 10% [vol/vol] glycerol, 0.2 mM PMSF) containing 0.1 M KCl
and 0.05% (vol/vol) NP-40, 100 µl of the DEAE-cellulose-bound
fraction was incubated with each antibody-bead complex for 5 h.
Immunoprecipitates were collected by centrifugation and washed four
times each with 1 ml of buffer E containing 0.4 M KCl and 0.05%
(vol/vol) NP-40. Protein complexes were eluted from the beads with 20 µl of Laemmli loading buffer (2% [wt/vol] sodium dodecyl sulfate
[SDS], 100 mM DTT, 60 mM Tris-HCl [pH 6.8], 0.001% [wt/vol]
bromophenol blue, 10% [vol/vol] glycerol) for Western blot analysis.
Affinity purification of CDK8-containing complexes.
Affinity
purification of CDK8-containing complexes was performed with antibodies
against CDK8. Affinity-purified anti-CDK8 antibodies were covalently
immobilized onto protein A-agarose beads ( Supercoiling reduction assay.
Plasmid chromatin was
assembled and purified as described previously (55). Each
reaction mixture (60 µl) contained chromatin (40 ng; assembled as
described in reference 55), topoisomerase I, and 4 mM ATP in buffer (4 mM MgCl2, 2 mM HEPES buffer [pH 7.5], 1 mM DTT, 20 mM KCl). The fraction used in the assay was the
affinity-purified CDK8 complex (20 µl of beads), control antibody
beads (20 µl), or highly purified Swi-Snf complex (Superose 12 fraction; 10 µl, 0.4 µg). Reaction mixtures were incubated for 30 min at 30°C, and reactions were terminated by the addition of 50 µl
of stop mixture (20 mM EDTA [pH 8.0], 0.2 M NaCl, 1% SDS, 0.25 mg of
glycogen per ml). Following treatment with proteinase K for 30 min at
37°C, the DNA was extracted with phenol-chloroform-isoamyl alcohol
(25:24:1), ethanol precipitated, and resolved by agarose gel
electrophoresis. The agarose gel was transferred to a nitrocellulose
membrane for Southern hybridization using a 32P-end-labeled
probe that hybridizes to the Histone acetyltransferase assay.
The assay was performed in
a 50-µl reaction volume containing 100 mM Tris-HCl buffer (pH 8.0),
8% (vol/vol) glycerol, 1 mM DTT, 1 mM PMSF, 10 µM butyric acid, and
0.1 mM EDTA with 130 nCi of [3H]acetyl-coenzyme A and 50 µg of calf thymus core histones and incubated for 30 min at 30°C.
Reaction products were spotted onto Whatman P-81 paper, air dried,
extensively washed in 50 mM sodium carbonate buffer (pH 9.1), and
dried. The amount of [3H]acetate incorporated was
determined by liquid scintillation counting.
Transcription assays.
Transcription reaction mixtures were
reconstituted as described previously (11). Transcription
factors were isolated as described in reference 41.
Protein interaction studies.
M2 affinity beads (Kodak; 10 µl) were incubated for 1 h at 4°C with SF9 cell extracts
containing baculovirus-expressed wild-type or truncated PCAF
(77) or p300 (53) polypeptides as indicated. The
beads were then washed extensively with 1 M KCl and 0.1% (vol/vol) NP-40 in buffer E. Prior to incubation with RNAPII, beads were equilibrated with buffer E containing 0.1 M KCl and 0.1% (vol/vol) NP-40. An RNAPII fraction (1.2 µg; Fig. 5A and B) containing
approximately equal amounts of the IIA and IIO forms was mixed with the
beads and incubated at 4°C for 1 h with rotation. The beads were
washed extensively with buffer E containing 0.1 M KCl and 0.1%
(vol/vol) NP-40. The immunoprecipitates were electrophoresed on a
5-to-20%-gradient SDS-polyacrylamide gel and analyzed by Western
blotting.
Resolution of two RNAPII complexes.
In HeLa cell nuclear
extracts, RNAPII exists in large-molecular-weight complexes (40,
56). We previously reported the purification of an RNAPII complex
that contains stoichiometric amounts of transcription factors IIE and
IIF, substoichiometric amounts of human Srb (hSrb) homologs (Srb7,
Srb10, and Srb11), DNA repair proteins, and limiting amounts of TFIIH
(11, 40). This complex can efficiently initiate
transcription upon the addition of TBP, TFIIB, and TFIIH.
Transcription reconstituted with TBP, TFIIB, TFIIH, and fractions
derived from a Sepharose CL-4B gel filtration column revealed the
presence of an RNAPII complex with a native molecular mass of
approximately 1.5 MDa (11) (Fig. 1A). Interestingly, Western blot analysis
using antibodies raised against the CTD of RNAPII revealed that a
substantial amount of RNAPII eluted with a native molecular mass
greater than 2 MDa (Fig. 1A). This RNAPII complex may be similar to the
larger RNAPII complex detected by glycerol gradient sedimentation
analysis in our previous study (40).
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Human RNA Polymerase II Complex Containing
Factors That Modify Chromatin Structure
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-mercaptoethanol, 10% [vol/vol] glycerol, 0.2 mM
phenylmethylsulfonyl fluoride [PMSF]) containing 0.1 M KCl (BC100)
(0.8 g of protein), 0.3 M KCl (0.5 g of protein), 0.5 M KCl (0.3 g of
protein), and 1.0 M KCl (0.1 g of protein). The 0.5 M KCl eluate was
dialyzed against BC100, and proteins were loaded onto a 50-ml
DEAE-cellulose (Whatman) column equilibrated with BC100. The
flowthrough fraction (also referred to as the 0.1 M KCl eluate;
0.1 g of protein) and the 0.35 M KCl eluates were collected. The
0.35 M KCl fraction (0.2 g of protein) was dialyzed against BC100 and
loaded onto a 40-ml S-Sepharose (Sigma) column equilibrated with BC100.
This column was developed with 20 column volumes of a linear gradient
from 0.1 to 0.8 M KCl in buffer C. Fractions containing RNAPII were
pooled and dialyzed against buffer C containing 500 mM KCl, 0.01%
(vol/vol) Triton X-100, 0.05% (vol/vol) Nonidet P-40 (NP40), and 20%
(vol/vol) glycerol. One-sixth of the pool was loaded onto a CL-4B
Sepharose (Pharmacia) gel filtration column (350 ml; 2.5 by 75 cm)
equilibrated in the same buffer. Fractions of 1.2 ml were collected and
analyzed by Western blot and transcription assays. Blue dextran
(Sigma), thyroglobulin (Sigma), and core RNAPII (41) were
loaded onto the same column as molecular weight markers. Fractions
containing Swi-Snf subunits were pooled, dialyzed against BC100, and
further chromatographed on a Mono S fast protein liquid chromatography 5/5 column (Pharmacia). The column was developed with a
10-column-volume linear gradient from 0.1 to 1.0 M KCl in buffer C. Every other fraction was analyzed by Western blotting and for histone
acetyltransferase activity.
1 mg/ml). Following
equilibration in buffer E containing 0.1 M KCl and 0.1% (vol/vol)
NP-40, antibody-cross-linked beads (0.5 ml) were incubated with the
DEAE-cellulose-bound fraction (0.35 M KCl eluate; 4 mg of protein) at
4°C with rotation. After incubation for 2 h, the beads were
washed extensively with buffer E containing 0.7 M KCl and 0.1%
(vol/vol) NP-40. Protein complexes were eluted from the column with 2.5 ml of 0.2 M glycine, pH 2.5, in five fractions. Forty microliters of
the glycine eluate was used in the silver staining analysis. The beads
were also used directly in functional assays.
-galactosidase gene in the backbone
plasmid (55). The DNA topoisomers were visualized by
autoradiography.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (33K):
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FIG. 1.
(A) Resolution of two different RNAPII complexes by
Sepharose CL-4B gel filtration chromatography. The upper panel
summarizes the Western blot results shown below. The lower panel shows
Western blots of fractions of the Sepharose CL-4B gel filtration
column. Blots were probed with antibodies as indicated at the side of
each blot. IIF (RAP74), the RAP74 subunit of TFIIF cycC, cyclin C. Input (1%; IN) and fraction numbers are indicated on the top of the
lower panel. The last row shows the transcription activity of the
column fractions obtained by using the adenovirus major late promoter
in a system reconstituted with TBP, TFIIB, and TFIIH. (B) The amount of
acetate incorporated into core histones and the protein concentration
of each Mono S (see Materials and Methods) column fraction. (C)
Coelution of CBP, PCAF, RNAPII (RPB1), BAF47, cyclin C, and CDK8 by
Mono S ion exchange chromatography. Fractions from a similar Mono S
column were analyzed by Western blotting using antibodies against CBP,
PCAF, RPB1, BAF47, cyclin C, and CDK8.
Coimmunoprecipitation of the Swi-Snf complex. The presence of the Swi-Snf complex in an RNAPII complex has been controversial, identified by one laboratory (75) but not by another (45). Therefore, we further investigated the putative association of this chromatin remodeling factor with the RNAPII holoenzyme by coimmunoprecipitation studies. Immunoprecipitation was performed under highly stringent conditions (0.4 M KCl, 0.05% NP-40) with antibodies against two hSrb proteins. Antibodies against hSrb10 (CDK8) and hSrb7 coimmunoprecipitated RNAPII, as expected (Fig. 2A). Importantly, these antibodies also immunoprecipitated the Swi-Snf complex as detected in a Western blot analysis using antibodies against the BAF47 subunit of the remodeling complex (Fig. 2A). The immunoprecipitation observed was specific, as control antibodies were not capable of coimmunoprecipitating any of the components of the RNAPII complex. This result is consistent with the coelution of RNAPII, hSrbs, and the Swi-Snf complex on multiple chromatographic steps as shown above. Although the stoichiometry of the Swi-Snf complex within the RNAPII complex has not been carefully analyzed, we believe, based on multiple chromatographic steps, that only a subpopulation of the Swi-Snf complex is associated with the RNAPII complex.
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Affinity purification of CDK8 (hSrb10)-cyclin C (hSrb11) containing the Swi-Snf complex. Because it is difficult to maintain the integrity of large-molecular-weight complexes during multiple chromatographic steps, we devised an affinity purification scheme to isolate the large RNAPII complex. Since our goal was to analyze whether the Swi-Snf complex was associated with the RNAPII complex by an approach different from that described above, antibodies to one of the Srb polypeptides, CDK8 (hSrb10), were used in the affinity purification procedure. The rationale behind choosing human Srb10 for the affinity purification step was based on the copurification and coimmunoprecipitation studies described above, as well as the regulation of the yeast SUC2 gene, which revealed a genetic interaction between Srb10 (and other components of the RNAPII holoenzyme complex) and the Swi-Snf complex (7, 66).
Interestingly, we observed that CDK8 (hSrb10) exists in multiple complexes in HeLa cell nuclear extracts (60). Upon fractionation of HeLa cell nuclear extracts on a phosphocellulose column, the majority of CDK8 and its regulatory subunit, cyclin C (hSrb11), eluted in the 0.1 to 0.3 and 0.3 to 0.5 M KCl fractions (Fig. 3A). The 0.3 to 0.5 M KCl eluate contains most of the GTFs as well as RNAPII and the Swi-Snf complex (Fig. 3A). Therefore, this material was further fractionated on a DEAE-cellulose column. Most of the RNAPII, GTFs, and the Swi-Snf complex bound to this column (Fig. 3A), whereas approximately half of the CDK8-cyclin C complex was found in the flowthrough fraction (see Materials and Methods and the legend to Fig. 3A). The DEAE-cellulose-bound fraction was used as the input sample for affinity chromatography. The affinity purification procedure isolated a CDK8 complex containing Swi-Snf subunits (BAF190, BAF170, BAF155, BAF60, and BAF47), cyclin C, hSrb7, and limiting amounts of RNAPII, as judged by Western blot analysis (Fig. 3B). This complex was specifically associated with CDK8, since none of these polypeptides were retained by the control antibody column (Fig. 3B, lane 4). This affinity-purified complex lacks GTFs (Fig. 3B and data not shown), reminiscent of the larger RNAPII complex purified by conventional chromatography. This was the case even under conditions where lower-concentration salt washes were used in the affinity purification step and RNAPII was more abundant in the complex (data not shown).
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The affinity-purified CDK8 complex remodels chromatin structure. We next analyzed whether the Swi-Snf complex present in the Srb10-containing complex was functional by a plasmid linking number reduction assay. This assay measures the reduction in plasmid linking number in the presence of topoisomerase I that results from a loss of nucleosomes or an alteration of nucleosome structure. The addition of a purified Swi-Snf complex to a chromatinized plasmid resulted in a gradual reduction in the superhelicity of the plasmid compared to that of a plasmid incubated with buffer alone (Fig. 4C, compare lanes 1 and 2). When the CDK8 affinity-purified complex was added to the assay mixture, a pattern similar to that observed with purified Swi-Snf complex was observed. The amounts of Swi-Snf added to the reaction mixtures were similar and were normalized by using BAF47 Western blot units. The supercoiling reduction activity observed was specific to the CDK8-containing complex, as the control antibodies (lane 3) treated in the same manner did not contain such activity. This result demonstrates that the CDK8-containing complex is active in chromatin remodeling.
The RNAPII complex contains histone acetyltransferase activity. Another activity that can increase chromatin accessibility is the modification of histones by acetylation. Previous studies have reported the presence of CBP within an RNAPII complex (47). Since the complex characterized above contains at least one nucleosome-remodeling activity, we investigated whether histone acetyltransferase activity was also present in the complex. Histone acetyltransferase activity coeluted with the large RNAPII complex in both the gel filtration (data not shown) and Mono-S (Fig. 1B) columns described above. We used Western blot analysis to identify the putative acetyltransferase polypeptides. We found that the histone acetyltransferases p300/CBP (the antibodies used in the Western blot analysis do not distinguish between CBP and p300) and PCAF (77) coeluted with RNAPII, Srb proteins, and the Swi-Snf complex (Fig. 1C). Another histone acetyltransferase, human Gcn5 (6, 77), that contains extensive homology with PCAF in the catalytic domain but that lacks the p300/CBP binding domain (77), was separated in earlier chromatographic steps and, therefore, was not present in the RNAPII complex (data not shown).
To further analyze the association of p300/CBP and PCAF with the RNAPII complex, we performed coimmunoprecipitation experiments using different antibodies. Antibodies against hSrb7 and hSrb10 (CDK8) specifically immunoprecipitated p300/CBP and PCAF (Fig. 2A). Using antibodies against the two histone acetyltransferases detected above, we observed a coimmunoprecipitation of RNAPII and Swi-Snf (Fig. 2B). The immunoprecipitation was specific because the control antibodies failed to immunoprecipitate any polypeptides. More importantly, antibodies against the CTD of the largest subunit of RNAPII immunoprecipitated RNAPII but abolished the coimmunoprecipitation of p300/CBP, PCAF, and Swi-Snf. This result suggested that these proteins are tethered, directly or indirectly, to the RNAPII complex through interactions involving the CTD which are disrupted (competed) by the anti-CTD antibodies.p300 and PCAF directly interact with different forms of RNAPII. Previous studies have indicated that CBP is a component of the human RNAPII complex and that its association with RNAPII is mediated by RHA (48). We sought to expand these studies to analyze the specificity and directness of the interaction between RNAPII and the p300/CBP-associated factor, PCAF, by coimmunoprecipitation experiments. We analyzed whether truncated and full-length PCAF proteins could interact with core RNAPII. More importantly, we sought to determine whether histone acetyltransferases p300 and PCAF could interact with both the initiation-competent nonphosphorylated and elongation-competent phosphorylated forms of RNAPII. Antibodies against the tag that is fused to the N terminus of each of the recombinant proteins were used in immunoprecipitation experiments with different forms of RNAPII. The silver staining of an SDS-polyacrylamide gel demonstrates the purity of the core RNAPII used in these experiments (Fig. 5A). Western blot analyses showed that only full-length PCAF can efficiently interact with RNAPII (Fig. 5C). Significantly, PCAF interacts with the phosphorylated, elongating form of RNAPII (Fig. 5C). In contrast to data published by others, we observed a direct interaction between RNAPII and p300 (Fig. 5D). The C terminus of p300 was sufficient for this direct interaction. Moreover, p300 directly and specifically immunoprecipitated the nonphosphorylated form of RNAPII. No interaction with the phosphorylated form of RNAPII could be detected (Fig. 5D). The interaction between p300 and RNAPII was not mediated by RHA, since the RNAPII used in these experiments was free of contaminating RHA as determined by silver staining (Fig. 5A) and Western blot analysis (Fig. 5B). However, our studies do not rule out the possibility that RHA can enhance the interaction between CBP and RNAPII. Our results demonstrate that PCAF and p300 can interact directly with RNAPII. Although PCAF does not interact with the initiating form of RNAPII, it is possible that PCAF is brought into the preinitiation complex through an interaction with p300.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have isolated an RNAPII complex containing factors that modify chromatin structure. Analysis of a gel filtration column reveals the existence of at least two different populations of RNAPII. The first elutes at approximately 1.5 MDa and contains GTFs and Srb homologs, while the second elutes at approximately 4 MDa and contains the Swi-Snf complex, Srb homologs, and histone acetyltransferases PCAF and p300/CBP. Whether these two RNAPII complexes are functionally distinct and carry out the transcription of different subsets of genes, whether they are species engaged in different steps of transcription, or whether they are subassemblies of a larger complex remains to be answered.
Three different RNAPII complexes have been isolated from yeast (7). Koleske and Young isolated the so-called RNAPII holoenzyme complex by using antibodies against different Srb polypeptides and Western blot analyses (31). This RNAPII complex contains the entire set of Srbs (Srb2 and Srb4 to Srb11) and most of the GTFs, except TBP and TFIIE. By contrast, Kim and coworkers (29) isolated the mediator complex by using a functional transcription assay analyzing for factors that mediate the response to transcription activation. The mediator is free of RNAPII and contains Srb2, Srb4 to Srb7, Rox3 (19), Gal11, Sin4, Rgr1 (37), and other polypeptides referred to as Meds (36, 45). This complex is devoid of Srb8 to Srb11. Interestingly, disruption of the SIN4 gene or a truncation of the RGR1 gene allowed the resolution of the mediator into two subcomplexes. One subcomplex is composed of Gal11, Sin4, Rgr1, and Med3 (37, 45), whereas the other subcomplex functionally resembles the mediator but lacks Gal11, Sin4, Rgr1, and Med3 (7). A third RNAPII complex has been described by Shi and coworkers (63). This complex appears to be functionally different from those described by Young and Kornberg as it lacks Srb and Med polypeptides but contains Paf1, Cdc73, Ccr1, and Hpr1 (8, 63).
The kinase-cyclin pair CDK8-cyclin C is the human homolog of the yeast Srb10-Srb11 complex (38). Previous studies have demonstrated the existence of different CDK8-cyclin C complexes in human cells (60). In agreement with these previous observations, we have chromatographically separated different CDK8-containing complexes. Immunoaffinity purification using antibodies against CDK8 demonstrated a tight association of the CDK8-cyclin C complex with the Swi-Snf complex as well as with hSrb7 and other polypeptides. This affinity-purified complex contains a substoichiometric amount of RNAPII. While this represents the first report of such a complex in mammalian cells, Wilson and coworkers demonstrated a physical and functional association between the Swi-Snf complex and the yeast RNAPII holoenzyme complex (75). However, apparently contradictory studies have been reported by Myers and coworkers, who found that the Swi-Snf complex is absent from a purified mediator complex (45). The studies of Carlson and coworkers analyzing the regulation of the SUC2 gene, together with our findings reported here may provide an explanation to the apparent paradox between the studies of Young and Kornberg. Neigeborn and Carlson identified the SNF family of genes based on their requirement for SUC2 expression (49). A subset of the SNF genes encode subunits of the Swi-Snf complex (SNF2, SNF5, SNF6, and SNF11), whereas SNF1 encodes a protein kinase. Components of the RNAPII holoenzyme complex were isolated as suppressors of snf1 mutations (SSN) (33, 66). The SSN genes encoding components of the RNAPII complex include SSN2 (SRB9), SSN3 (SRB10), SSN4 (SIN4), SSN5 (SRB8), SSN7 (ROX3), and SSN8 (SRB11) and are negative regulators of transcription (7, 20). Kornberg and colleagues isolated the mediator complex as an activity that mediates the response to activators by using a functional reconstituted transcription system. Therefore, this may have resulted in the purification of a complex lacking the negative regulator Srb8 to Srb11. The RNAPII complex isolated by Young and coworkers was purified by using antibodies against different Srb polypeptides in Western blot analyses. The findings presented here suggest that the Swi-Snf complex is tethered to the RNAPII complex through interactions mediated by the CDK8-cyclin C-containing complex. Thus, it is possible that the RNAPII complex is a dynamic entity composed of subcomplexes. One subcomplex is the mediator, which is involved in positive regulation; another subcomplex is composed of Sin4, Rgr1, Gal11, and Med3 (37), and another subcomplex includes Srb8 to Srb11 (7). Our studies suggest that the Swi-Snf complex is tethered to the RNAPII complex via an Srb10-Srb11-containing complex. Importantly, we have recently isolated a CDK8-containing complex similar to the one described in these studies but devoid of the Swi-Snf polypeptides. This complex was derived from the DEAE-cellulose flowthrough fraction described in the legend for Fig. 3A and functions to inhibit the response to transcriptional activators (67a). However, a large population of Swi-Snf complexes appears to be devoid of CDK8, judged by coimmunoprecipitation and chromatographic studies.
An important point that must be addressed is the relative abundance of the different RNAPII complexes in the cell. This is difficult to address experimentally, because upon cell disruption the majority of RNAPII (>90%) is found in the insoluble nuclear pellet (41). Extraction of RNAPII from this fraction requires sonication in high-ionic-strength buffer, which disrupts any large RNAPII complexes. Indeed, we and others have previously purified the core RNAPII from this insoluble protein fraction (39, 41, 61). By contrast, we find that the small proportion of RNAPII that remains in the soluble protein fraction upon cell disruption (the nuclear extract) is associated with other proteins in high-molecular-weight complexes (40). We have thus far purified two such RNAPII complexes, one containing GTFs and the other lacking GTFs, but containing the Swi-Snf complex. Based on yields from purification, we estimate that the former complex is more abundant. We also note that the complex we have purified by immunoaffinity chromatography using CDK8 antibodies contains only a small amount of RNAPII. This observation is in agreement with those of Kornberg and colleagues, who isolated a mediator complex containing a limiting amount of RNAPII (15, 27, 28, 45).
The large RNAPII complex was also found to contain two or three histone acetyltransferases, p300 and/or CBP and PCAF. CBP and PCAF have been previously shown to interact with multiple transcriptional activators in various coactivator complexes. During multiple chromatographic procedures, we observed a large population of CBP and PCAF in other complexes. Whether these complexes contain RNAPII or other components of the RNAPII complex has not been determined. Interestingly, thus far no yeast RNAPII complex has been shown to contain histone acetyltransferase activity. In yeast, GCN5, a component of a coactivator complex (adapter), contains histone acetyltransferase activity. GCN5 exists in multimeric complexes termed ADA (17, 42) and SAGA (Spt-Ada-Gcn5 acetyltransferase) (17), which have been separated from the RNAPII complex. Two human Gcn5 homologs, hGcn5 and PCAF, have been isolated. Human Gcn5 was found not to cofractionate with the human RNAPII complexes. There are other nuclear histone acetyltransferases in yeast (59), and it is therefore possible that some may exist in association with the RNAPII holoenzyme complex. In support of this idea are recent experiments demonstrating that recruitment of the RNAPII holoenzyme complex to the PHO5 promoter is sufficient for the displacement of four positioned nucleosomes (16). Importantly, expression of the PHO5 promoter is independent of the Swi-Snf chromatin remodeling complex (16). Therefore, displacement of the four nucleosomes is unlikely to be mediated by the Swi-Snf complex, and it is possible that the RNAPII complex includes histone acetyltransferases or other nucleosome remodeling activities. The actual mechanism remains to be elucidated. These experiments demonstrate, however, that recruitment of the RNAPII holoenzyme complex is sufficient to remove four positioned nucleosomes at the PHO5 promoter in vivo and reveals a functional association of the RNAPII complex with chromatin remodeling activities.
In these studies we have also analyzed the nature of the interaction between RNAPII and the histone acetyltransferases. Our studies revealed that p300 and PCAF interact directly with RNAPII. Using a recombinant glutathione S-transferase-CBP truncated polypeptide, others observed a requirement for RHA for the interaction between CBP and RNAPII (48). The discrepancy between the two studies may be due to the different truncated proteins used. The studies of others used a smaller version of CBP, and it is possible that the RNAPII-interacting domain was removed. However, in support of the studies of Nakajimo and coworkers (48), we have found RHA in our RNAPII complexes (data not shown).
Importantly, we found that each of the acetyltransferases interacts with a different form of RNAPII. p300 was found to interact with the nonphosphorylated form of RNAPII, which is the initiation-competent form of the enzyme. By contrast, PCAF was found to interact with the phosphorylated, elongation-competent form of RNAPII. This suggests that the RNAPII complex may be loaded onto the promoter through interactions mediated by p300 or CBP. These two polypeptides have been shown to interact, in addition to RNAPII, with GTFs, a large number of different sequence-specific DNA-binding proteins, and coactivators (25, 64, 73). p300 or CBP or both are common components of different signal transduction pathways (25). It is possible that upon "activation" of p300/CBP, this large polypeptide interacts with the RNAPII complex. This newly formed complex would contain sequence-specific DNA-binding proteins (those interacting with p300/CBP) and would recruit chromatin remodeling activity and histone acetyltransferase activity to specific genes thereby facilitating DNA binding. Once the full preinitiation complex is formed, RNAPII can initiate transcription. Upon phosphorylation of the CTD, the interactions between RNAPII and factors are disrupted. Only interactions that tolerate this transition or that interact specifically with the phosphorylated form of RNAPII will remain associated with the elongating polymerase (67). This transition may allow PCAF to be transferred from p300/CBP to RNAPII during elongation where it can acetylate nucleosomal histones along the body of the gene.
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ACKNOWLEDGMENTS |
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We thank Edio Maldonado for thoughtful comments and advice during early steps of the work. We also thank G. LeRoy for purifying the Swi-Snf complex, W. Wang and G. Crabtree for human Swi-Snf subunit antibodies, M. Montminy for the CBP antibodies, J. Hurwitz for the RNA antibodies, R. Young for the hSRB7 antibodies, and members of the Reinberg laboratory for helpful suggestions. We also thank Mike Hampsey for helpful comments on the manuscript.
This work was supported by a grant from the National Institutes of Health (GM-37120) and from the Howard Hughes Medical Institute to D.R.
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ADDENDUM |
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Results similar to those reported here were reported by Neish et al. (49a) following submission of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Howard Hughes Medical Institute, Division of Nucleic Acid Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 663 Hoes Ln., Piscataway, NJ 08854-5635. Phone: (732) 235-4195. Fax: (732) 235-5294. E-mail: reinbedf{at}umdnj.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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