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Molecular and Cellular Biology, June 2001, p. 3876-3887, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3876-3887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

E. Tory Manning,1 Tsuyoshi Ikehara,2 Takashi Ito,2 James T. Kadonaga,3 and W. Lee Kraus1,4,*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 148531; Department of Biochemistry, Saitama Medical School, Morohongo, Iruma-gun, Saitama 350-0495, Japan2; Section of Molecular Biology, University of California, San Diego, La Jolla, California 92093-03473; and Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 100214

Received 7 November 2000/Returned for modification 22 December 2000/Accepted 16 March 2001

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.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, Cornell University, 465 Biotechnology Building, Ithaca, NY 14853. Phone: (607) 255-6087. Fax: (607) 255-6249. E-mail: wlk5{at}cornell.edu.

Molecular and Cellular Biology, June 2001, p. 3876-3887, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3876-3887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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