Biochemical Analysis of Distinct Activation Functions in p300 That Enhance Transcription Initiation with Chromatin Templates

Abstract

To investigate the mechanisms of transcriptional enhancement by the p300 coactivator, we analyzed wild-type and mutant versions of p300 with a chromatin transcription system in vitro. Estrogen receptor, NF-κB p65 plus Sp1, and Gal4-VP16 were used as different sequence-specific activators. The CH3 domain (or E1A-binding region) was found to be essential for the function of each of the activators tested. The bromodomain was also observed to be generally important for p300 coactivator activity, though to a lesser extent than the CH3 domain/E1A-binding region. The acetyltransferase activity and the C-terminal region (containing the steroid receptor coactivator/p160-binding region and the glutamine-rich region) were each found to be important for activation by estrogen receptor but not for that by Gal4-VP16. The N-terminal region of p300, which had been previously found to interact with nuclear hormone receptors, was not seen to be required for any of the activators, including estrogen receptor. Single-round transcription experiments revealed that the functionally important subregions of p300 contribute to its ability to promote the assembly of transcription initiation complexes. In addition, the acetyltransferase activity of p300 was observed to be distinct from the broadly essential activation function of the CH3 domain/E1A-binding region. These results indicate that specific regions of p300 possess distinct activation functions that are differentially required to enhance the assembly of transcription initiation complexes. Interestingly, with the estrogen receptor, four distinct regions of p300 each have an essential role in the transcription activation process. These data exemplify a situation in which a network of multiple activation functions is required to achieve gene transcription.

ACKNOWLEDGMENTS

We thank John Lis, Patricia Willy, Steve Nordeen, Dmitry Fyodorov, Mark Levenstein, and Paul Mason for critical reading of the manuscript. We are grateful to John Hiscott for the p65 baculovirus, Marc Montminy for the GST-E1A fusions, Jessica Tyler for advice on the AT assays, Yoshihiro Nakatani for the FLAG-E1A baculovirus, Benita Katzenellenbogen for the 2ERE-pS2-CAT construct, and Y. Cha Henderson, A. Deisseroth, and T. Burke for the hIRF-1 constructs.

This work was supported by a grant from the National Institutes of Health (GM46995) to J.T.K. W.L.K. was supported by a postdoctoral fellowship from the American Cancer Society, California Division, and by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund.