Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the protein CREB, which is activated as a result of phosphorylation by protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by protein kinase A binds specifically to a nuclear protein of M(r) 265K which we term CBP (for CREB-binding protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric protein to function as a protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.

The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.

The Snf-2-related CREB-binding protein activator protein (SRCAP) serves as a coactivator for a number of transcription factors known to interact with CBP. Swr1, the closest Saccharomyces cerevisiae ortholog of SRCAP, is a component of the chromatin remodeling complex SWR-C, which catalyzes exchange of the histone variant H2A.Z into nucleosomes. In this report, we use a combination of conventional chromatography and anti-SRCAP immunoaffinity chromatography to purify a native human SRCAP complex with a polypeptide composition similar to that of SWR-C, and we show for the first time that this SRCAP-containing complex supports ATP-dependent exchange of histone dimers containing H2B and H2A.Z into mononucleosomes reconstituted with recombinant H2A, H2B, H3, and H4. Our findings, together with previous evidence implicating H2A.Z in transcriptional regulation, suggest that SRCAP's coactivator function may depend on its ability to promote incorporation of H2A.Z into chromatin.

The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Here, we show that activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength-dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras-MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription.

Studies in Saccharomyces cerevisiae indicate the histone variant H2A.Z is deposited at promoters by the chromatin remodeling protein Swr1 and plays a critical role in the regulation of transcription. In higher eukaryotes, however, little is known about the distribution, method of deposition, and function of H2A.Z at promoters. Using biochemical studies, we demonstrated previously that SRCAP (SNF-2-related CREB-binding protein activator protein), the human ortholog of Swr1, could catalyze deposition of H2A.Z into nucleosomes. To address whether SRCAP directs H2A.Z deposition in vivo, promoters targeted by SRCAP were identified by a chromatin immunoprecipitation (ChIP)-on-chip assay. ChIP assays on a subset of these promoters confirmed the presence of SRCAP on inactive and active promoters. The highest levels of SRCAP were observed on the active SP-1, G3BP, and FAD synthetase promoters. Detailed analyses of these promoters indicate sites of SRCAP binding overlap or occur adjacent to the sites of H2A.Z deposition. Knockdown of SRCAP levels using siRNA resulted in loss of SRCAP at these promoters, decreased deposition of H2A.Z and acetylated H2A.Z, and a decrease in levels of SP-1, G3BP, and FAD synthetase mRNA. Thus, these studies provide the first evidence that SRCAP is recruited to promoters and is critical for the deposition of H2A.Z.Chromatin remodeling has emerged as a key mechanism for gene regulation in development and cancer. The histone variant H2A.Z is a universally conserved intrinsic component of eukaryotic chromatin (1). Studies in Saccharomyces cerevisiae indicate that H2A.Z is required for normal gene expression, is distributed throughout the genome, and appears to be required for proper recruitment of RNA polymerase II (RNAP II) 2 and TATA-binding protein (TBP) (2). The highest levels of H2A.Z in S. cerevisiae occur within nucleosomes located at inactive promoters where it has been postulated to provide the correct promoter architecture to facilitate activation of transcription (3-5). Activation of transcription results in decreased levels of H2A.Z and an increase in acetylated H2A.Z, which has been proposed to facilitate disassembly/reassembly of nucleosomes (6 -8).In higher eukaryotes, the genomic distribution and the biological function(s) of H2A.Z are poorly defined. In mammals, H2A.Z is essential for embryonic development and chromosome segregation, and increased H2A.Z expression is implicated in cardiac hypertrophy (9 -11). Studies done in chicken cells suggest that deposition of both H2A.Z and acetylated H2A.Z in higher eukaryotes differs from that observed in S. cerevisiae and occurs at active promoters but not at inactive promoters (12, 13). The specific role that H2A.Z plays at active promoters in higher eukaryotes has not been established.The exchange of H2A.Z into nucleosomes in S. cerevisiae has been demonstrated by genetic and biochemical approaches to be carried out by the catalytic subunit of the SWR-C complex, termed Swr1 (14, 15). A SRCAP complex, which is the human ortholog of the...

CREB-binding protein (CBP) functions as a coactivator molecule for a number of transcription factors including CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. Although binding sites for these factors within CBP have been identified, the regions of CBP responsible for transcriptional activation are unknown. In this report, we show that the N-terminal half of CBP is sufficient for activation of CREB-mediated transcription and that this region contains a strong transcriptional activation domain (TAD). Both deletion of this TAD or sequestering of factors that the TAD binds using a squelching assay were found to greatly decrease the ability of CBP to activate CREB-mediated transcription. In vivo studies by others have shown that p300/CBP associates with TBP; using an in vitro approach, we show the N-terminal TAD binds TBP. We also examined the ability of the C terminus of CBP to activate transcription using GAL-CBP chimeras. With this approach, we identified two C-terminal TADs located adjacent to the c-Fos binding site. In previous studies, cAMP-dependent protein kinase A (PKA) increased the transcriptional activity of a GAL full-length CBP chimera in F9 cells, and of the C terminus in PC-12 cells. Here, we demonstrate that PKA also increased the ability of the N-terminal TADs of CBP to activate transcription in PC-12 but not F9 or COS-7 cells, suggesting that this PKA-responsiveness is cell type-specific.Cyclic AMP induces transcription of a number of genes through activation of the members of the cAMP response element-binding protein (CREB) 1 family of transcription factors (1, 2), which interact with a conserved promoter element termed the CRE (cAMP response element) (3-6). CREB activates basal transcription through several domains that bind basal transcription factors (TFIIB, TATA-binding protein (TBP), and TAF II 110) (7, 8) and activates cAMP-responsive transcription through a separate region termed the kinaseinducible domain (9). Serine 133 within the kinase-inducible domain is phosphorylated by a number of kinases including cyclic AMP-dependent protein kinase (PKA), and this phosphorylation promotes interaction of CREB with a second nuclear factor, CREB-binding protein (CBP) (10). This interaction is critical for activation of transcription, since antibodies that block formation of a CBP⅐CREB complex prevent cAMP-responsive transcription (11). In contrast, increasing the amount of CBP available to interact with CREB enhances PKA-activated CREB-mediated transcription by up to 6-fold (12). CBP fused to a heterologous DNA binding domain can directly activate transcription, suggesting that PKA-phosphorylated CREB provides a scaffold for recruitment of CBP that activates transcription (10).While PKA phosphorylation of CREB is required for association of CREB with CBP, several studies indicate it is not sufficient for activation of transcription (13). This requires additional PKA-mediated events, which may include modification of the activity of CBP, since PKA enhances the ability of a GAL4-CBP chimera t...

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.

The ability of cAMP response-element binding protein (CREB)-binding protein (CBP) to function as a co-activator for a number of transcription factors appears to be mediated by its ability to act as a histone acetyltransferase and through its interaction with a number of other proteins (general transcription factors, histone acetyltransferases, and other co-activators). Here we report that CBP also interacts with a novel ATPase termed Snf2-Related CBP Activator Protein (SRCAP). Consistent with this activity, SRCAP contains the conserved ATPase domain found within members of the Snf2 family. Transfection experiments demonstrate that SRCAP is able to activate transcription when expressed as a Gal-SRCAP chimera and that SRCAP also enhances the ability of CBP to activate transcription. The adenoviral protein E1A was found to disrupt interaction between SRCAP and CBP possibly representing a mechanism for E1A-mediated transcriptional repression. CREB 1 -binding protein (CBP) has been found to function as a co-activator for a growing number of sequence specific transcription factors including CREB, the STATs, and the nuclear receptors (1-5). Binding studies have identified several regions of CBP that interact with general transcription factors such as TBP, TFIIB, and RNAP II (2, 6 -8), suggesting it functions as a co-activator in part by recruiting these proteins to the promoter. CBP has also been shown to have intrinsic histone acetyltransferase (HAT) activity and to bind to several proteins with HAT activity (P/CAF, ACTR, NCoA-1). This suggests that CBP alone, or acting in conjunction with these proteins, functions as a co-activator by stimulating remodeling of chromatin (9 -12). This is supported by the work of Korus et al. (13) who demonstrate that several transcription factors have a specific requirement for the HAT activity of NCoAs, P/CAF, and CBP for activation of transcription. The adenoviral protein E1A also binds CBP but represses the ability of CBP to function as a co-activator for CREB as well as a number of other transcription factors (4,5,14,15). This appears to be due in part to the ability of E1A to prevent binding of P/CAF and P/CIP to the C-terminal end of CBP. E1A also binds to the N-terminal end of CBP and suppresses the ability of a Gal-CBP-(1-450) chimera to activate transcription. Although P/CAF also binds to this same region, competition between P/CAF and E1A binding has not been demonstrated (5).Deletion of amino acids 1-460 abolishes the ability of CBP to serve as a co-activator for CREB and STAT-1 but not for other transcription factors such as the retinoic acid receptor (5, 6). In "squelching-type" assays, overexpression of CBP amino acids 1-460 has also been found to block the ability of full-length CBP to activate CREB-mediated transcription (5). Studies from several laboratories indicate that this region of CBP contacts proteins, including TBP and P/CAF, which may be involved in the activation of transcription (5, 6). Microinjection studies support such a role for P/CAF by demonstrating ...