Adaptor-mediated Recruitment of RNA Polymerase II to a Signal-dependent Activator

Abstract

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 CBP⅐RNA polymerase II complex is critical for expression of cAMP-responsive genes.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 g) (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 (Ϫ56 to Ϫ32). Affinity-purified antisera were added to in vitro transcription assays as reported (5).Immunoprecipitation Assays-For immunoprecipitation assays, HeLa nuclear extract (100 g) 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 ␣ 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 incuba...

Protein Kinase A Regulatory Subunit Type IIβ Directly Interacts with and Suppresses CREB Transcriptional Activity in Activated T Cells

“…Phosphorylation of Ser133 promotes recruitment of the coactivator CREB-binding protein (CBP) or its paralog p300. CBP/p300 mediates gene transcription via its association with the basal transcription machinery of the RNA polymerase II holoenzyme complex (21) and its intrinsic histone acetyltransferase activity (22). However, Ser133 phosphorylation is necessary but not sufficient to mediate CREB transactivation.…”

p300 functions as a transcriptional coactivator for the TAL1/SCL oncoprotein

“…They have a recognized function as transcriptional coactivators for diverse families of transcription factors, including the nuclear steroid receptors (Chakravarti et al, 1996;Hanstein et al, 1996;Kamei et al, 1996;Yao et al, 1996), and bHLH Mutoh et al, 1998;Qiu et al, 1998;Sartorelli et al, 1997;Yuan et al, 1996), leucine zipper (Bannister and Kouzarides, 1995;Bannister et al, 1995;Chrivia et al, 1993;Mink et al, 1997), and zinc ®nger (Blobel et al, 1998;Lee et al, 1995) proteins. Acting as transcriptional adaptors, they link sequence-speci®c DNA-binding proteins to components of the basal transcriptional machinery (Abraham et al, 1993;Kee et al, 1996;Nakajima et al, 1997) and to proteins with histone acetyltransferase activity (Yang et al, 1996). Acetylation of core histones in turn induces a localized disruption in nucleosome structure, which facilitates access of transcription factors to DNA (reviewed in Shikama et al, 1997).…”

Involvement of Co-activator p300 in the Transcriptional Regulation of the HER-2/neu Gene

“…7). p300/CBP is capable of binding to either the enhancer-binding proteins (8,9) or the proteins involved in basal transcriptional machinery including the TATA-binding protein or TBP (10) and the RNA polymerase II (11). p300/CBP serves as an adaptor that bridges the enhancer-specific factors to the basal transcriptional apparatus consequently transactivating gene.…”