(from the application): The long-term objective of this project is to understand the relation between chromatin structure and transcriptional regulation. There is a complex array of factors at the interface of chromatin and transcription, and researchers are now faced with the challenge of learning how they work. This grant proposes to use a biochemical approach to the analysis of chromatin and transcription to provide a better understanding of how the noncovalent alteration and covalent modification of chromatin affect nucleosome mobility and transcriptional activity.
The specific aims are as follows. 1. Investigate the biochemical function of transcription factors with chromatin templates assembled with purified factors. The cloning of the chromatin assembly factor, ACF, has led to the achievement of a purified recombinant chromatin assembly system.
This aim i nvolves the use of the purified chromatin assembly system for the analysis of nucleosome mobility and transcriptional regulation. 2. Investigate the basis of transcriptional regulation by p300 and other coactivators with chromatin templates (assembled with either the S190 extract or the purified assembly factors).
The aim of these studies is to investigate how transcriptional coactivators regulate transcriptional activity in the natural context of a chromatin template. 3. Examine the effects of histone structure and modification upon nucleosome mobility and transcriptional activity of chromatin templates in vitro.
This aim will investigate the effects of specific patterns of histone acetylation upon nucleosome mobility and transcriptional activation by using unmodified bacterially-synthesized histones in conjunction with histone-modifying enzymes. In addition, experiments will test whether histones are the key downstream targets of histone acetyltransferases (HATs) and histone deacetylases (HDACs) by the generation and analysis of mutant recombinant histones. These studies should contribute to our fundamental understanding of gene expression and should be applicable to the analysis of human diseases that might be treated by the specific control of transcription factors or chromatin.
