ABSTRACT
The RNA polymerase II (Pol II) holoenzyme in yeast is an essential transcriptional regulatory complex which has been defined by genetic and biochemical approaches. The mammalian counterpart to this complex, however, is less well defined. Experiments herein demonstrate that, along with Pol II and SRB proteins, proteins associated with transcriptional regulation as cofactors are associated with the Pol II holoenzyme. Earlier experiments have demonstrated that the breast cancer-associated tumor suppressor BRCA1 and the CREB binding protein (CBP) were associated with the holoenzyme complex. The protein related to CBP, the E1A-associated p300 protein, is shown in these experiments to be associated with the holoenzyme complex as well as the BRG1 subunit of the chromatin remodeling SWI/SNF complex. Importantly, the Pol II holoenzyme complex does not contain some factors previously reported as stoichiometric components of the holoenzyme complex, most notably the proteins which function in repair of damaged DNA, such as PCNA, RFC and RPA. The presence of the p300 coactivator and the chromatin-modifying BRG1 protein support a role for the Pol II holoenzyme as a key target for regulation by enhancer binding proteins.
The biochemical characterization of factors required for transcription of mRNA in eukaryotic cells has revealed a requirement for RNA polymerase II (Pol II) plus the basal transcription factors TFIIB, -D, -E, -F and -H (1,2). Regulation of the transcription reaction additionally requires coactivator proteins which link these basal factors to the enhancer binding regulatory factors. Complementary genetic and biochemical approaches in yeast have shown that Pol II, basal factors and coactivators exist as a pre-assembled complex termed the Pol II holoenzyme, an entity which is highly responsive to regulation by DNA binding transcriptional activators (3-5). The holoenzyme was initially defined as a Pol II-containing complex associated with a class of proteins called SRB factors (suppressors of RNA polymerase B mutations). Nine SRB factors were originally identified in a yeast genetic screen as mutants which can reverse a Pol II mutation and which form a complex physically bound to the C-terminal domain (CTD) of Pol II (6). Pol II plus these SRB subunits are obligate components of the Pol II holoenzyme. Recruitment of the Pol II holoenzyme to a promoter is sufficient for activation of a test gene (7,8) and it has been shown that a temperature-sensitive mutation in SRB4 results in complete loss of transcription at the restrictive temperature (9), suggesting that the holoenzyme form of Pol II is responsible for virtually all mRNA transcription initiation in the yeast cell.
In mammalian cells the counterpart of the yeast holoenzyme has only recently been described. Mammalian homologs of three of the nine yeast SRB subunits have been identified based on sequence homology (10-13) and, similar to the yeast complex, are found stably associated with Pol II (10, 11). Additionally, the human SRB7 protein complemented a partial defect in yeast SRB7 (10), demonstrating conservation of function.
Multiple protocols for purification of the mammalian Pol II holoenzyme have been described. These include single step purification via an anti-cdk7 antibody from liver nuclear extract (14), a multistep purification of an SRB7-containing complex from calf thymus extracts (10), two chromatographic steps from a HeLa nuclear extract followed by an anti-TFIIF monoclonal antibody affinity matrix step (11,15), a single step purification from HeLa whole cell extract on a TFIIS affinity matrix (16) or a purification, developed by our laboratory, in which Pol II holoenzyme is purified from HeLa whole cell extract in two to three steps (17,18). This last preparation technique yields holoenzyme which is responsive to appropriate regulation in vitro by the phospho-CREB transcriptional activator (18).
These disparate purification protocols do not produce an identical complement of protein species co-purifying as the Pol II holoenzyme. In one study CBP failed to co-purify with the Pol II holoenzyme (16), while in another study CBP in the holoenzyme complex was the critical component for activation of transcription in vitro (18). In one report the factors which repair damaged DNA were identified as stable components of the Pol II holoenzyme and were present in the complex in amounts stoichiometric relative to the polymerase (11). In the experiments described here we demonstrate that the proteins which regulate repair of damaged DNA are not present in the holoenzyme complex, that the CBP homolog p300 is a component of the Pol II holoenzyme and that the BRG1 subunit of the SWI/SNF complex is in the holoenzyme complex.
HeLa S3 cells or HeLa cells carrying epitope-tagged TBP (19) were passaged in suspension culture using standard procedures. Whole cell extracts were prepared using the technique of Manley et al. (20), except that all buffers containing chloride were replaced with buffers containing acetate. Purification of this whole cell extract over Biorex70 ion exchange matrix, centrifugation of the 0.6 M KOAc protein fraction over sucrose gradients and chromatographic purification of the sucrose gradient peak over a Ni-NTA matrix have been described (17).
For preparative sucrose gradients 6 ml Biorex70 0.6 M peak were applied to the sucrose gradients. For analysis of sensitivity to nucleases 0.6 ml Biorex70 0.6 M peak fraction were treated with 25 µg RNase A (DNase-free) at 20°C for 30 min and then layered directly onto the sucrose gradient without terminating the reaction. Alternatively, 10 µg DNase I were incubated with 0.6 ml Biorex70 0.6 M peak fraction in 10 mM Mg(OAc)2 and 2 mM CaCl2 at 20°C for 30 min and the reaction terminated by addition of EGTA to 2 mM final concentration and EDTA to 15 mM final concentration. The proteins were then layered onto the sucrose gradient as above. For ethidium bromide analysis all sucrose solutions and the sample had ethidium bromide added to a final concentration of 1 µg/ml and centrifuged as above.
The glutathione S-transferase (GST) fusion protein encoding the holoenzyme binding domain of CBP (amino acid residues 1805-1890; a kind gift of T.Nakajima and M.Montminy; 18) was expressed in bacteria [BL21(DE3)]. One liter of induced bacterial culture was extracted using standard procedures and lysate was bound to 1 ml glutathione-agarose. This matrix was then washed extensively with 0.8 M KOAc in H buffer (20 mM Tris-OAc, 1 mM EDTA, 20% glycerol, 1 mM PMSF and 2 mM DTT). The concentration of the CBP polypeptide fragment on the matrix was 0.3 mg/ml matrix.
For analysis by immunoblot of Pol II holoenzyme polypeptides which bound to the CBP matrix 2 µg GST-CBP(1805-1890), equal amounts of GST fusion protein alone and GST-CBP(1-117) were bound to 25 µl glutathione-agarose beads and incubated with peak protein fraction from the Biorex70 0.6 M wash (25 µl, 75 µg total protein diluted in 0.5 ml buffer H with KOAc adjusted to 0.15 M, 0.5% NP-40 and BSA added to 0.2 mg/ml) at 4°C for 16 h. The beads were washed three times with 0.5 ml buffer H + 0.4 M KOAc. Bound proteins were eluted in buffer containing SDS and 2-mercaptoethanol for analysis by SDS-PAGE and immunoblotting. Immunoblotting was done as per standard procedures.
BRCA1 C-terminal amino acids 1560-1863 were fused to the biotin binding protein of the PinPoint Xa-3 vector (Promega) and in TG1 bacterial cells expressed a fusion protein of ~45 kDa. A BRCA1 domain identical to the protein described here except for a point mutation from methionine to glutamate at amino acid residue 1775 was also fused to the PinPoint vector. These fusion proteins and the PinPoint domain alone were purified on magnetic beads containing streptavidin (Dynal) and the amounts of fusion protein bound to the matrix were normalized to each other by analysis on SDS-PAGE, electrotransfer of the proteins to nitrocellulose and probing with HRP-conjugated streptavidin (Gibco-BRL). Equal amounts of immobilized fusion protein were incubated for 16 h at 4°C with 25 µl (75 µg) Biorex70 0.6 M peak fraction diluted to 0.5 ml in buffer H + 0.15 M KOAc, plus 0.2 mg/ml BSA, 0.1 mM DTT and 0.5% NP-40. The matrix was extensively washed in buffer H + 0.6 M KOAc, 0.2 mg/ml BSA, 1 mM DTT and 0.5% NP-40 and bound sample subjected to SDS-PAGE and immunoblotting by standard procedures.
This purification strategy was similar to that used in the experiments which described the co-purification of CBP and of BRCA1 (17,18). For the purpose of identifying fractions the Pol II holoenzyme was operationally defined as containing both Pol II and the SRB10/11 homologs cdk8 and cycC. No other assumptions were made about composition of the Pol II holoenzyme. An advantage of the Biorex70 matrix is that most cellular proteins flow through (Neish et al., submitted for publication), but, as will be shown below, many transcription factors bind. As can be seen in the Western blots depicted in Figure 1, about half of Pol II and nearly all of cycC eluted in the 0.6 M step elution from the Biorex70 column. These data were consistent with the co-elution profile of cdk8, BRCA1 and half of the Pol II reported earlier (17).
It was possible that the entity we defined as the Pol II holoenzyme complex was in fact composed of proteins which were not physically associated, but rather linked via DNA or RNA molecules. For example, a Pol II molecule elongating along a DNA template and synthesizing a nascent mRNA might appear to bind to other factors via the DNA or RNA molecules. This possibility was tested by treating the input sample for the sucrose gradient with RNase A or DNase I or by running the entire sucrose gradient in ethidium bromide (Fig. 4 and data not shown). If a polypeptide was associated with the Pol II holoenzyme via a DNA or RNA tether then one of these treatments would cause it to sediment at the top of the gradient and no longer in the holoenzyme peak fractions. Aliquots of the same input Biorex70 fraction were treated with RNase or DNase and then run simultaneously in four identical sucrose gradients in order to improve comparability of results. As can be seen in Figure 4A, the control/untreated gradient was similar to that in Figure 2. Pol II peaked in fractions 9-17 in this centrifugation run. The p300 protein did not have a significant pool in the low sedimentation samples but was reproducibly seen in pools accumulating in fractions 1-3 and 11-17. Both cdk8 and cycC co-sedimented in fractions 9-13 and were present in lower concentrations in fraction 15. These results were typical of many repeated centrifugation runs in which cdk8 and cycC were in higher concentration in the more rapidly sedimenting portion of the holoenzyme peak. BRCA1 protein was seen in two main pools, one coincident with holoenzyme fractions 9-15 and one pool in fractions 1-3. The SWI/SNF subunit BRG1 was in multiple fractions, including a pool in fractions 1-3 and a broad peak from 9-19 overlapping with the holoenzyme peak fractions.
When the input sample was treated with RNase A prior to centrifugation no effect was observed in the holoenzyme pool in fractions 9-15 (Fig. 4B). All of the tested factors were again present in these fractions. The Pol II and BRG1 polypeptides did extend into more slowly sedimenting fractions, as observed in the control gradient. Clearly, the complexes in fractions 9-15 were unaffected by the treatment. Strikingly, the pool of material in fractions 1-3 containing p300, BRCA1 and BRG1 disappeared, suggesting that the high sedimentation rate complex was assembled on an RNA molecule.
The DNase I and ethidium bromide treatments yielded results which were largely comparable with the control sample (data not shown). We concluded from these analyses that although BRCA1, BRG1 and p300 could be associated with RNA, co-purification of these proteins with Pol II and the SRB factors as a complex in the middle of the gradient was not a consequence of their interaction with DNA or RNA.
It had been noted that a Pol II complex physically interacted with a CBP domain spanning amino acid residues 1805-1890 (18). The CBP(1805-1890) protein was tested as an affinity matrix to determine whether it could purify the other putative holoenzyme subunits along with Pol II. The CBP matrix specifically bound to Pol II, as previously reported, and also retained p300, CBP and BRCA1 (Fig. 5B). GST or GST-CBP(1-117) control matrices did not bind any of these proteins. Purification of holoenzyme containing p300 and CBP on the CBP affinity matrix suggested that multiple binding sites for CBP/p300 exist in the holoenzyme complex. Since the CBP matrix could interact with the holoenzyme, some of these binding sites were unoccupied.
Identical results were obtained whether the input protein source was the Biorex70 0.6 M fraction or Pol II holoenzyme fractions from the sucrose gradient (data not shown). Thus BRCA1, Pol II and CBP/p300 exist as a size-selected complex which was bound to the CBP affinity matrix. Affinity purification of Pol II on CBP matrix was unaffected by RNase digestion (data not shown).
The RPA proteins, which did not co-purify with the holoenzyme on conventional chromatography, did not bind to the CBP affinity matrix (Fig. 5B). The Biorex70 column fraction input contained both the Pol II holoenzyme and the RPA proteins, but the RPA proteins did not bind to this affinity matrix. These results supported the conclusion that the proteins involved in repair of damaged DNA were not components of the Pol II holoenzyme complex being purified in this study.
The SWI/SNF subunit BRG1, a fraction of which co-purified with the holoenzyme (Figs 1-3) and which was in a complex containing hSRB7 (Fig. 3B), was not purified significantly on the CBP matrix. This result suggests that the SWI/SNF subunit is only in a small subpopulation of the Pol II holoenzyme. As will be shown below, a similar matrix containing BRCA1 specifically purifies BRG1, suggesting that this SWI/SNF component may be associated with certain subpopulations of the Pol II holoenzyme.
The C-terminus of BRCA1 (residues 1560-1863) has been shown to function as a transcriptional activator when expressed in eukaryotic cells as a fusion protein with the GAL4 DNA binding domain (24). When the methionine at codon 1775 was mutated to glutamate this transcriptional activation was lost (24). The wild-type BRCA1 fragment and the M1775E mutant were fused to the biotin binding PinPoint protein and used as affinity matrices. When these matrices were incubated with the Biorex70 0.6 M fraction the wild-type BRCA1 matrix purified Pol II, while the M1775E mutation failed to purify the polymerase (Fig. 6B). Washing of the matrix in these assays was quite stringent (0.6 M KOAc and 0.5% NP-40 in buffer H). The stringency of the wash protocol and the failure of a protein containing a single point mutation to purify the holoenzyme suggested that this assay was quite specific for holoenzyme components. These results suggest that the failure of this M1775E mutant BRCA1 protein to activate transcription in a transfection assay (24) might be explained by a failure to bind to the Pol II holoenzyme.
It was found that the wild-type BRCA1 matrix purified the BRG1 subunit of the SWI/SNF complex (Fig. 6B, bottom panel). It had been shown that a significant amount of BRG1 was in a complex with the Pol II holoenzyme (Figs 1-3) but that very little bound to the CBP affinity matrix. BRG1 was purified by the wild-type BRCA1 matrix but not the M1775E matrix, indicating that the interaction with this SWI/SNF subunit was specific.
Purification of the Pol II holoenzyme in these experiments revealed that p300 and BRG1, but not DNA repair factors, are components of the holoenzyme complex. The specificity of the purification protocol is demonstrated by these latter proteins, which are abundant in the nucleus and which do not co-purify with the holoenzyme complex.
The protein composition of the Pol II holoenzyme preparation was tested by several rigorous criteria. Pol II holoenzyme sedimentation characteristics were not affected by treatments which would perturb protein-nucleic acid interactions. Strikingly, RNase A treatment of the sucrose gradient input material did not affect sedimentation of holoenzyme peak fractions but did cause disappearance of BRCA1, p300 and BRG1 from fractions with very high sedimentation rates (fractions 1-3; Fig. 4). This pool of BRCA1 had been the second major pool of BRCA1 in the cell extract (17) and this result with RNase A treatment suggests that virtually all of the BRCA1 in the cell extract is associated with the holoenzyme. It is possible that this RNase-sensitive complex represents the Pol II holoenzyme with attached nascent transcript and this possibility is currently being tested.
We thank E.Nigg, P.Rickert, E.Lees, R.Scully, R.Eckner, D.M.Livingston, R.E.Kingston, G.Schnitzler, R.Young and A.Dutta for the kind gift of antibody reagents. We also thank R.Scully, A.Monteiro, H.Hanafusa, T.Nakajima, M.Montminy and Z.Burton for expression plasmids used in this study. We thank A.Dutta and M.Montminy for helpful advice during the course of this research. This work was supported in part by NIH grant GM53504 and a Junior Faculty Research Award from the American Cancer Society to J.D.P., a KO8 award HL03011-04 from the NIH to A.S.N. and an institutional training grant T32HL07627-11 from the NIH to S.F.A.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Purification of Pol II holoenzyme and basal transcription factors
Nuclease treatment of samples for sucrose sedimentation analysis
Affinity purification of the Pol II holoenzyme with the CBP matrix
Affinity purification of the Pol II holoenzyme with the BRCA1 matrix
Results
Purification strategy
Co-sedimentation of holoenzyme components was not dependent upon nucleic acids
CBP affinity matrix
BRCA1 affinity matrix
Discussion
Acknowledgements
References
REFERENCES
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S. Y. Wu, M. C. Thomas, S. Y. Hou, V. Likhite, and C. M. Chiang Isolation of Mouse TFIID and Functional Characterization of TBP and TFIID in Mediating Estrogen Receptor and Chromatin Transcription J. Biol. Chem., August 13, 1999; 274(33): 23480 - 23490. [Abstract] [Full Text] [PDF]
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H. Kimura, Y. Tao, R. G. Roeder, and P. R. Cook Quantitation of RNA Polymerase II and Its Transcription Factors in an HeLa Cell: Little Soluble Holoenzyme but Significant Amounts of Polymerases Attached to the Nuclear Substructure Mol. Cell. Biol., August 1, 1999; 19(8): 5383 - 5392. [Abstract] [Full Text] [PDF]
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C. Sune and M. A. Garcia-Blanco Transcriptional Cofactor CA150 Regulates RNA Polymerase II Elongation in a TATA-Box-Dependent Manner Mol. Cell. Biol., July 1, 1999; 19(7): 4719 - 4728. [Abstract] [Full Text] [PDF]
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K. Ellwood, W. Huang, R. Johnson, and M. Carey Multiple Layers of Cooperativity Regulate Enhanceosome-Responsive RNA Polymerase II Transcription Complex Assembly Mol. Cell. Biol., April 1, 1999; 19(4): 2613 - 2623. [Abstract] [Full Text] [PDF]
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Y.-F. Hu, Z. L. Hao, and R. Li Chromatin remodeling and activation of chromosomal DNA replication by an acidic transcriptional activation domain from BRCA1 Genes & Dev., March 15, 1999; 13(6): 637 - 642. [Abstract] [Full Text]
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D. T. Haile and J. D. Parvin Activation of Transcription in Vitro by the BRCA1 Carboxyl-terminal Domain J. Biol. Chem., January 22, 1999; 274(4): 2113 - 2117. [Abstract] [Full Text] [PDF]
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F. C. P. Holstege and R. A. Young Transcriptional regulation: Contending with complexity PNAS, January 5, 1999; 96(1): 2 - 4. [Full Text] [PDF]
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V. E. Myer and R. A. Young RNA Polymerase II Holoenzymes and Subcomplexes J. Biol. Chem., October 23, 1998; 273(43): 27757 - 27760. [Full Text] [PDF]
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X.-B. Qiu, Y.-L. Lin, K. C. Thome, P. Pian, B. P. Schlegel, S. Weremowicz, J. D. Parvin, and A. Dutta An Eukaryotic RuvB-like Protein (RUVBL1) Essential for Growth J. Biol. Chem., October 23, 1998; 273(43): 27786 - 27793. [Abstract] [Full Text] [PDF]
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T. K. Kim, T. H. Kim, and T. Maniatis Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro PNAS, October 13, 1998; 95(21): 12191 - 12196. [Abstract] [Full Text] [PDF]
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H. Cho, G. Orphanides, X. Sun, X.-J. Yang, V. Ogryzko, E. Lees, Y. Nakatani, and D. Reinberg A Human RNA Polymerase II Complex Containing Factors That Modify Chromatin Structure Mol. Cell. Biol., September 1, 1998; 18(9): 5355 - 5363. [Abstract] [Full Text]
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O Papoulas, S. Beek, S. Moseley, C. McCallum, M Sarte, A Shearn, and J. Tamkun The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes Development, January 10, 1998; 125(20): 3955 - 3966. [Abstract] [PDF]
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W. Zhang, D. E. Geiman, J. M. Shields, D. T. Dang, C. S. Mahatan, K. H. Kaestner, J. R. Biggs, A. S. Kraft, and V. W. Yang The Gut-enriched Kruppel-like Factor (Kruppel-like Factor 4) Mediates the Transactivating Effect of p53 on the p21WAF1/Cip1 Promoter J. Biol. Chem., June 9, 2000; 275(24): 18391 - 18398. [Abstract] [Full Text] [PDF]
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Y.-F. Hu, T. Miyake, Q. Ye, and R. Li Characterization of a Novel Trans-Activation Domain of BRCA1 That Functions in Concert with the BRCA1 C-terminal (BRCT) Domain J. Biol. Chem., December 22, 2000; 275(52): 40910 - 40915. [Abstract] [Full Text] [PDF]
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T. Miyake, Y.-F. Hu, D. S. Yu, and R. Li A Functional Comparison of BRCA1 C-terminal Domains in Transcription Activation and Chromatin Remodeling J. Biol. Chem., December 15, 2000; 275(51): 40169 - 40173. [Abstract] [Full Text] [PDF]
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B. P. Schlegel, V. J. Green, J. A. A. Ladias, and J. D. Parvin BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription PNAS, March 28, 2000; 97(7): 3148 - 3153. [Abstract] [Full Text] [PDF]
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T. Ouchi, S. W. Lee, M. Ouchi, S. A. Aaronson, and C. M. Horvath Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-gamma target genes PNAS, May 9, 2000; 97(10): 5208 - 5213. [Abstract] [Full Text] [PDF]
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