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Molecular and Cellular Biology, December 1999, p. 8123-8135, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

W. Lee Kraus,dagger E. Tory Manning, and James T. Kadonaga*

Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0347

Received 21 July 1999/Returned for modification 2 September 1999/Accepted 9 September 1999

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-kappa B 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.

* Corresponding author. Mailing address: Department of Biology, 0347, Pacific Hall, Room 2212B, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0347. Phone: (858) 534-4608. Fax: (858) 534-0555. E-mail: jkadonaga{at}ucsd.edu.

dagger Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853.

Molecular and Cellular Biology, December 1999, p. 8123-8135, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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