Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro

Abstract

In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

We thank A. Vojtek for providing plasmids and the yeast strain L40 and M. Deckert for valuable discussions and help with confocal laser microscopy. We thank Carl Wu for ISWI cDNA and Y. Nakatani (Dana Farber) for recombinant p300 protein. We also thank Y. Ishimi and J. Kadonaga for the gift of NAP-1 antiserum. We are indebted to David Chambers for help with fluorescence-activated cell sorter analysis.

This work was supported by National Institutes of Health grants and by grants from the Japanese Society for the Promotion of Science (H.A.) and the Research for the Future Program (T.I.).

H.A. is on sabbatical leave from Okayama University Medical School. S.T.-D. is an investigator of Institut National de la Santé et de la Recherche Médicale (INSERM). T.H. is a Frank and Else Schilling American Cancer Society Research Professor.

Hiroshi Asahara, Sophie Tartare-Deckert, Takeya Nakagawa, and Tsuyoshi Ikehara contributed equally to this work.