©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Adaptor-mediated Recruitment of RNA Polymerase II to a Signal-dependent Activator (*)

(Received for publication, October 31, 1995; and in revised form, November 28, 1995)

Barbara L. Kee (§) Jonathan Arias Marc R. Montminy (¶)

From the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The second messenger cAMP stimulates the expression of a number of target genes via the protein kinase A-mediated phosphorylation of CREB at Ser-133 (Gonzalez, G. A., and Montminy, M. R.(1989) Cell 59, 675-680). Ser-133 phosphorylation enhances CREB activity by promoting interaction with a 265-kDa CREB binding protein referred to as CBP (Arias, J., Alberts, A., Brindle, P., Claret, F., Smeal, T., Karin, M., Feramisco, J., and Montminy, M.(1994) Nature 370, 226-228; Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H.(1993) Nature 365, 855-859). The mechanism by which CBP in turn mediates induction of cAMP-responsive genes is unknown but is thought to involve recruitment of basal transcription factors to the promoter. Here we demonstrate that CBP associates specifically with RNA polymerase II in HeLa nuclear extracts. This association in turn permits RNA polymerase II to be recruited to CREB in a phospho-(Ser-133)-dependent manner. As anti-CBP antiserum, which inhibits recruitment of CBP and RNA polymerase II to phospho-(Ser-133) CREB, attenuates transcriptional induction by protein kinase A in vitro, our results demonstrate that the CBPbulletRNA polymerase II complex is critical for expression of cAMP-responsive genes.


INTRODUCTION

A number of hormones and growth factors stimulate the expression of target genes by inducing the reversible phosphorylation of specific transcription factors(4) . Although phosphorylation has been shown to regulate a number of nuclear factors by inducing their nuclear targeting or DNA binding activities, the cAMP-responsive transcription factor CREB belongs to a group whose transactivation potential is affected(1, 5) . In this regard, Chrivia et al.(3) have characterized a nuclear CREB binding protein, termed CBP, which binds to CREB in a phospho-(Ser-133)-dependent manner. The requirement for CBP in mediating cAMP-dependent transcription has been demonstrated by cellular microinjection experiments in which CBP antisera blocked transcriptional induction by cAMP (2) and by transient transfection experiments in which overexpression of CBP could potentiate CREB activity in response to agonist(2, 6) . Here we examine the mechanism by which CBP interacts with the transcriptional apparatus to induce target gene expression in response to hormonal stimulation. Our results suggest that CBP is constitutively associated with specific components of the transcriptional apparatus and that this association in turn permits recruitment of certain basal factors to promoters of cAMP-responsive genes.


EXPERIMENTAL PROCEDURES

Preparation of Nuclear Extracts and in Vitro Transcription Assays

Nuclear extract preparations and in vitro transcription assays were carried out as described previously(5) . To evaluate the effect of PKA (^1)on in vitro transcription reactions, purified recombinant PKA catalytic subunit (1 µg) (kindly provided by S. Taylor) was added to HeLa nuclear extracts during transcription reactions. CREB activity was monitored with an adenovirus major late promoter template containing three cAMP-responsive elements (CREs) from the rat somatostatin promoter (-56 to -32). Affinity-purified antisera were added to in vitro transcription assays as reported(5) .

Immunoprecipitation Assays

For immunoprecipitation assays, HeLa nuclear extract (100 µg) was precleared with protein A-Sepharose for 30 min at 4 °C. Precleared extract was incubated with primary antibody for 1 h at 4 °C. To detect alpha RNA polymerase II, a monoclonal antiserum, raised against a C-terminal domain polypeptide of the large subunit (Promega), was used. Antibody complexes were recovered by incubation with protein A-Sepharose beads for 1 h at 4 °C. Beads were washed three times with buffer (B100) containing 100 mM KCl, 10 mM Tris, 1% Nonidet P-40, resuspended in 2 times SDS loading buffer, and analyzed by SDS-polyacrylamide gel electrophoresis.

Glutathione-Sepharose Chromatography

A CREB cDNA fragment encoding the kinase-inducible domain (aa 88-160) was fused in-frame to the glutathione S-transferase (GST) cDNA in the pGEX-2T plasmid (Promega). GSTbulletKID fusion protein was expressed, and purification from BL21 Escherichia coli was carried out as described previously(3) . GSTbulletKID fusion protein was phosphorylated at Ser-133 using purified PKA catalytic subunit as described previously(7) . For GST affinity chromatography, HeLa extracts (100 µg) were added to glutathione-Sepharose beads (50 µl) containing GSTbulletKID or GST phospho-(Ser-133)bulletKID polypeptides and co-incubated for 1 h at 4 °C. Beads were washed three times with B100 buffer (see above) and then evaluated by Western blot analysis.


RESULTS AND DISCUSSION

Preliminary evidence suggesting that CBP migrates as a high molecular mass complex of 2000 kDa during gel filtration chromatography (not shown) prompted us to examine whether CBP might stimulate cAMP-responsive genes by virtue of its association with specific basal transcription factors. When purified from HeLa nuclear extracts by phosphocellulose chromatography (Fig. 1, top), CBP was detected predominantly in the 0.3 M KCl ``B'' fraction, which also contained RNA polymerase II. Following subsequent fractionation over a Mono-S ion exchange resin (Fig. 1, bottom), CBP again eluted with peak fractions of RNA polymerase II. In contrast to the relatively sharp elution profile for CBP, however, RNA polymerase II appeared to be more broadly distributed, indicating that only a fraction of RNA polymerase II may be associated with CBP. By contrast with RNA polymerase II, CBP did not co-elute from the Mono-S column with TFIIB, a basal factor that has been reported to interact with CBP in GST pull-down assays(6) .


Figure 1: The CREB co-activator CBP fractionates with RNA polymerase II during purification from HeLa nuclear extracts. Top, Western blot analysis of HeLa nuclear extract following fractionation over P11 phosphocellulose resin. A-D correspond to HeLa protein fractions eluted from the P11 column with 100 mM, 300 mM, 500 mM, and 1.0 M KCl, respectively. Analysis of CBP, RNA polymerase II (RNA Pol II) in individual fractions as indicated on left. Bottom, Western blot analysis of the 300 mM KCl HeLa ``B'' fraction (top) following fractionation over Mono-S ion exchange resin. Elution profile for CBP and RNA polymerase II as indicated on left. FT, flow-through.



To test whether CBP in fact associates with RNA polymerase II, we performed co-immunoprecipitation studies with two distinct anti-CBP antisera directed against aa 1-100(5729) and aa 455-679(5614) of the protein (Fig. 2A). Although neither antiserum was capable of recognizing purified RNA polymerase II directly by Western blot or immunoprecipitation assay (not shown), the large subunit of RNA polymerase II was detected in immunoprecipitates of HeLa extracts with both antisera under non-denaturing conditions. In agreement with the broad elution profile of RNA polymerase II following Mono-S chromatography, only a limited fraction (10-20%) of the RNA polymerase II large subunit appeared to be associated with CBP in HeLa extracts. By contrast with RNA polymerase II, other basal transcription factors such as TBP and TFIIB did not appear to associate detectably with CBP in co-fractionation or co-immunoprecipitation assays (Fig. 1), indicating that the CBP-RNA polymerase II interaction was indeed specific. In this regard, RNA polymerase II was found to co-precipitate with CBP even at high concentrations of KCl (0.8 M), suggesting that this complex was also stable.


Figure 2: CBP is associated with RNA polymerase II in HeLa nuclear extracts. A, Western blot analysis of immunoprecipitates (IP) from HeLa nuclear extracts using anti-CBP antiserum (alphaCBP) and anti-RNA polymerase II antibody (alphaRNA POL II). Antisera used for immunoprecipitation are indicated over each lane. PI, preimmune antiserum; 5729, rabbit polyclonal alphaCBP antiserum raised against CBP polypeptides containing aa 1-117; 5614, alphaCBP antiserum raised against CBP recombinant CBP polypeptide extending from aa 455-679; POLII, monoclonal alphaRNA polymerase II antiserum raised against a C-terminal domain polypeptide (Promega); ONPUT, crude HeLa nuclear extract prior to immunoprecipitation. Relative mass, in kilodaltons, indicated alongside. Asterisks indicate position of CBP and RNA polymerase II large subunit. Additional CBP-immunoreactive bands in lanes marked 5614 and 5729 correspond to proteolytic digestion products of CBP, which are generated during immunoprecipitation with those antisera. B, CBP mediates recruitment of RNA polymerase II to CREB following PKA-dependent phosphorylation at Ser-133. Western blot analysis of HeLa nuclear extracts following affinity chromatography on glutathione-Sepharose resins containing unphosphorylated (GSTbulletKID) or Ser-133 phosphorylated (GST-(P)-KID) GST-CREB polypeptide. ONPUT, HeLa nuclear extract (100 µg) before pull-down assay; MW, relative mass (in kilodaltons). alphaCBP, anti-CBP antiserum; alphaRNA POL II, anti-RNA polymerase II antiserum.



Previous reports showing that CBP interacts with a KID (aa 88-160) in CREB (2, 3) prompted us to examine whether CBP mediates the PKA-dependent recruitment of RNA polymerase II to this region (Fig. 2B). Following affinity chromatography of crude HeLa nuclear extracts over glutathione-Sepharose resin containing either GSTbulletKID or GST-phospho-(Ser-133)bulletKID fusion proteins, CBP was specifically bound to phospho-(Ser-133)bulletKID resin. Similarly, RNA polymerase II was detected on resins containing phospho-(Ser-133)bulletKID but not unphosphorylated KID peptide. As purified RNA polymerase II was unable to bind to phospho-(Ser-133)bulletKID directly (not shown), these results indicate that RNA polymerase II is recruited to phospho-(Ser-133)bulletKID via CBP.

To test whether PKA simulates formation of a heteromeric complex consisting of phospho-(Ser-133) CREBbulletCBPbulletRNA polymerase II, as predicted by GST affinity chromatography experiments, we performed immunoprecipitation assays on crude HeLa nuclear extracts (Fig. 3). Using a CREB antiserum (253) that can recognize the CREBbulletCBP complex(8) , we detected CBP in immunoprecipitates from PKA-treated but not untreated HeLa nuclear extracts. Similarly, the large subunit of RNA polymerase II was recovered specifically from immunoprecipitates of PKA-treated HeLa extracts, demonstrating that PKA induces formation of a phospho(Ser-133) CREBbulletCBPbulletRNA polymerase II complex.


Figure 3: PKA stimulates formation of a phospho-(Ser-133) CREB-CBP-RNA polymerase II (POLII) heteromeric complex in HeLa nuclear extracts. Western blot analysis (alphaCBP, alphaRNA POLII) of immunoprecipitates (IP) prepared from control(-) or PKA (+) treated HeLa nuclear extract with various antisera under non-denaturing conditions. ONPUT, crude HeLa nuclear extract prior to immunoprecipitation; 253, polyclonal anti-CREB antiserum raised against the full-length recombinant CREB protein (aa 1-341); 5729, CBP antiserum raised against a recombinant CBP polypeptide extending from aa 1-100; PI, preimmune serum.



In order to determine whether recruitment of the CBPbulletpolymerase II complex to phospho-(Ser-133) CREB is critical for transcriptional induction by PKA, we performed in vitro transcription assays on crude HeLa nuclear extracts (Fig. 4). Addition of PKA to nuclear extracts induced transcription from a cAMP-responsive template containing three consensus cAMP-responsive elements (3 times CRE) approximately 4-fold. But PKA treatment had no effect on an internal control adenovirus major late promoter template lacking CRE sites. Addition of affinity-purified CBP antiserum, which blocks recruitment of the CBPbulletRNA polymerase II complex to CREB(2) , specifically inhibited PKA-inducible transcription from the 3 times CRE template. Unrelated antiserum (anti-corticotropin-releasing factor binding protein) had no effect on PKA induction of the 3 times CRE template, however, demonstrating that the inhibition by anti-CBP antiserum was indeed specific.


Figure 4: The CBP-RNA polymerase II complex is required for transcriptional induction by PKA in vitro. Primer extension analysis of in vitro transcription reactions performed with HeLa nuclear extracts in the presence or absence of purified PKA catalytic subunit, as indicated above each lane (-, +). 3XCRE, transcription template containing three tandemly repeated somatostatin CRE sites inserted upstream of the adenovirus major late promoter. MLP, adenovirus major late promoter construct lacking CRE sequences. Transcription reactions containing affinity-purified alphaCBP antiserum or control anti-corticotropin-releasing factor binding protein (alphaCRFBP) antiserum as indicated over corresponding lanes.



Our observation that CBP is found in a high molecular weight complex is supported by recent findings of Maldonado et al.,(^2)suggesting that CBP is contained within purified preparations of a mammalian RNA polymerase II holoenzyme. In contrast to results of Kwok et al.(6) , TFIIB did not appear to associate detectably with CBP in HeLa extracts. These results would suggest that TFIIB may interact with CBP only after being recruited to the promoter.

Our results do not address whether CBP interacts directly with RNA polymerase II. In preliminary GST pull-down assays, purified recombinant CBP polypeptides are unable to associate directly with the large subunit of RNA polymerase II, (^3)suggesting that the interaction between CBP and RNA polymerase II may either require post-translational modification (i.e. phosphorylation) or may involve other proteins within the RNA polymerase II holoenzyme complex.

In a previous study, we found that the activity of purified phospho-(Ser-133) CREB in cell-free transcription assays was indistinguishable from that of unphosphorylated CREB(9) . In this report, we found the addition of PKA to HeLa nuclear extracts during the transcription reaction was critical for Ser-133 phosphorylation-dependent activity. These results are consistent with recent findings that other PKA-dependent events in addition to CREB phosphorylation are required for transcriptional induction by cAMP in vivo(8) . In this regard, CBP contains a consensus PKA phosphorylation site at Ser-1772, and it is tempting to speculate that the interaction between CBP and RNA polymerase II may itself be regulated by CBP phosphorylation. In yeast, interaction between an upstream activator (GAL4) and a component of the yeast RNA polymerase II holoenzyme complex (GAL11) is sufficient for transcriptional induction(10) . The importance of CBP in mediating not only cAMP but also mitogen-inducible transcription (2) indicates that CBP may similarly provide contact points for recruitment of mammalian RNA polymerase II holoenzyme by multiple signal-dependent activators.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 37828 and was funded in part by The Foundation for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Medical Research Council of Canada.

To whom correspondence should be addressed: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-453-4100 (ext. 1502); Fax: 619-552-1546.

(^1)
The abbreviations used are: PKA, protein kinase A; CRE, cAMP-responsive element; aa, amino acids; GST, glutathione S-transferase; KID, kinase-inducible domain.

(^2)
E. Maldonado and D. Reinberg, personal communication.

(^3)
B. L. Kee and M. Montminy, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. D. Reinberg for sharing information about the polymerase II holoenzyme complex prior to publication. We thank Dr. K. Ferreri and other members of the Montminy laboratory for helpful discussions.


REFERENCES

  1. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59, 675-680 [Medline] [Order article via Infotrieve]
  2. Arias, J., Alberts, A., Brindle, P., Claret, F., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994) Nature 370, 226-228 [CrossRef][Medline] [Order article via Infotrieve]
  3. Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-859 [CrossRef][Medline] [Order article via Infotrieve]
  4. Hunter, T., and Karin, M. (1992) Cell 70, 375-387 [Medline] [Order article via Infotrieve]
  5. Gonzalez, G. A., Menzel, P., Leonard, J., Fischer, W. H., and Montminy, M. R. (1991) Mol. Cell. Biol. 11, 1306-1312 [Medline] [Order article via Infotrieve]
  6. Kwok, R., Lundblad, J., Chrivia, J., Richards, J., Bachinger, H., Brennan, R., Roberts, S., Green, M., and Goodman, R. (1994) Nature 370, 223-225 [CrossRef][Medline] [Order article via Infotrieve]
  7. Hagiwara, M., Brindle, P., Harootunian, A., Armstrong, R., Rivier, J., Vale, W., Tsien, R., and Montminy, M. R. (1993) Mol. Cell. Biol. 13, 4852-4859 [Abstract]
  8. Brindle, P., Nakajima, T., and Montminy, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10521-10525 [Abstract]
  9. Alberts, A., Arias, J., Hagiwara, M., Montminy, M., and Feramisco, J. (1994) J. Biol. Chem. 269, 7623-7630 [Abstract/Free Full Text]
  10. Barberis, A., Pearlberg, J., Simkovich, N., Farrell, S., Reinagel, P., Bamdad, C., Sigal, G., and Ptashne, M. (1995) Cell 81, 359-368 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.