title: 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, template-committed 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.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Manning
authors.forename: T
authors.initials: T
authors.lastname: Ikehara
authors.forename: T
authors.initials: T
authors.lastname: Ito
authors.forename: J T
authors.initials: JT
authors.lastname: Kadonaga
authors.forename: W L
authors.initials: WL
authors.lastname: Kraus
mesh_terms: D000818:Animals; D001665:Binding Sites; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila; D004355:Drug Stability; D006657:Histones / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D002843:Chromatin; D006657:Histones; D019476:Insect Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D015534:Trans-Activators