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

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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, 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 versionsof p300 with a chromatin transcription system in vitro. Estrogenreceptor, NF- $kappa$ B p65 plus Sp1, and Gal4-VP16 were used as differentsequence-specific activators. The CH3 domain (or E1A-binding region)was found to be essential for the function of each of the activatorstested. The bromodomain was also observed to be generally importantfor p300 coactivator activity, though to a lesser extent thanthe CH3 domain/E1A-binding region. The acetyltransferase activityand the C-terminal region (containing the steroid receptor coactivator/p160-bindingregion and the glutamine-rich region) were each found to be importantfor activation by estrogen receptor but not for that by Gal4-VP16.The N-terminal region of p300, which had been previously foundto interact with nuclear hormone receptors, was not seen to berequired for any of the activators, including estrogen receptor.Single-round transcription experiments revealed that the functionallyimportant subregions of p300 contribute to its ability to promotethe assembly of transcription initiation complexes. In addition,the acetyltransferase activity of p300 was observed to be distinctfrom the broadly essential activation function of the CH3 domain/E1A-bindingregion. These results indicate that specific regions of p300 possessdistinct activation functions that are differentially requiredto enhance the assembly of transcription initiation complexes.Interestingly, with the estrogen receptor, four distinct regionsof p300 each have an essential role in the transcription activationprocess. These data exemplify a situation in which a network ofmultiple activation functions is required to achieve genetranscription.

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

Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY14853.

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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