p300 Forms a Stable, Template-Committed Complex with Chromatin: Role for the Bromodomain

Abstract

The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable, templatecommitted complex with chromatin during the transcription process. The template commitment is dependent on the time of incubation of p300 with the chromatin template and occurs independently of the presence of a transcriptional activator protein. In studies examining interactions between p300 and chromatin, we found that p300 binds directly to chromatin and that the binding requires the p300 bromodomain, a conserved 110-amino-acid sequence found in many chromatin-associated proteins. Furthermore, we observed that the isolated p300 bromodomain binds directly to histones, preferentially to histone H3. However, the isolated p300 bromodomain does not bind to nucleosomal histones under the same assay conditions, suggesting that free histones and nucleosomal histones are not equivalent as binding substrates. Collectively, our results suggest that the stable association of p300 with chromatin is mediated, at least in part, by the bromodomain and is critically important for p300 function. Furthermore, our results suggest a model for p300 function that involves distinct activator-dependent targeting and activator-independent chromatin binding activities.Activated transcription of genes by RNA polymerase II (RNA Pol II) requires the concerted actions of sequencespecific DNA-binding transcriptional activators, chromatin remodeling complexes, histone acetyltransferases (HATs), and coactivators. Together, these factors function to overcome the transcriptional repression caused by the packaging of genes into chromatin and to stimulate the activity of RNA Pol II. The initiating event in the process of activated transcription is the binding of transcriptional activator proteins to binding sites in the promoters of target genes. In many cases, the activities of the activator proteins are regulated by input from cellular signaling pathways (e.g., steroid hormones and mitogen-activated protein kinase pathways). Once bound to DNA, activators can recruit chromatin remodeling complexes, HATs, and coactivators to the promoter, leading to the formation of a stable RNA Pol II preinitiation complex and, subsequently, transcription initiation (reviewed in references 3, 19, 23, 30, 34, 50, and 67).Transcriptional coactivators are a diverse group of factors and multipolypeptide complexes, some of which possess intrinsic HAT activity (16,42,43,45,63,68). Coactivators with HAT activity include p300 and the closely related CREB binding protein (CBP; often referred to with p300 as p300/CBP) (4, 20), as well as the p300/CBP-associated factor (PCAF), which functions as part of a multipolypeptide complex (54, 69). In general, coact...

p300/Cyclic AMP-Responsive Element Binding-Binding Protein Mediates Transcriptional Coactivation by the CD28 T Cell Costimulatory Receptor

“…The C-terminal portion of the protein contains intact HAT and transactivation domains that have inducible coactivator activity in Jurkat T cells (44). However, it lacks a bromodomain thought important for binding to chromatin (45). In contrast, a Gal4-CBP 1-450 N-terminal fragment strongly transactivated in the absence of any stimulation (Fig.…”

Roles of CREB-binding protein (CBP)/p300 in respiratory epithelium tumorigenesis

“…Mechanistically, histone acetylation promotes the accessibility of DNA to transcription protein complexes, by facilitating the "unwiring" of the chromatin structure [16]. CBP/p300 can interact with chromatin nucleosomes via nucleosome assembly proteins, histone-binding proteins and possibly histones themselves [17,18]. In addition to histones, CBP/p300 also modulate a variety of other proteins by acetylation [19,20].…”

The CBP Bromodomain and Nucleosome Targeting Are Required for Zta-Directed Nucleosome Acetylation and Transcription Activation

“…The p300 bromodomain, which is most closely related to the CBP bromodomain, was shown to be important for transcription initiation and responsiveness to estrogen receptor activation in vitro (35). The p300 bromodomain was also found to bind to chromatin and was essential for a stable, template-committed transcription complex (38). The bromodomains of Swi2 and GCN5 were also found to be required for anchoring of these chromatin-modifying complexes to acetylated chromatin templates (29).…”

“…We deleted the CBP bromodomain and E1A interacting region of the C/H3 domain. The bromodomain has been implicated in chromatin (38) and acetyllysine binding (17,29,31,54), and the C/H3 domain is the target of numerous protein interactions, including contacts with Zta (55). Consistent with previous findings, full-length CBP modestly stimulated Zta activation of the BRLF1 promoter (Fig.…”