The long-range objective of our proposed research is to determine how phosphorylation of histones H3 and H1 affects chromatin structure and how these histone modifications might contribute to leukemias and other oncogenic disease. Phosphorylation of histones H3 and H1 is a hallmark of rapidly proliferating cells, and the levels of these modified histones are induced upon cellular transformation and vary as a function of cell cycle and cell growth condition. The sites for H3 and H1 phosphorylation are contained within positive charged domains that are known to play crucial roles in the higher order folding of nucleosomal arrays; phosphorylation has been hypothesized to lead to a decondensation of chromatin that might be more permissive for transcription. In addition, we propose that the phosphorylation of histones H3 and H1 might facilitate the ability of ATP-dependent chromatin """"""""remodeling"""""""" factors to recognize their target chromosomal loci and facilitate nucleosome disruption. This proposal will use a combination of biochemical and cell biological approaches to directly test the role of H3 ane H1 phosphorylation in the structure of the nucleosome, the folding of model nucleosomal arrays, and in the recruitment and activity of the SWI/SNF family of ATP-dependent chromatin remodeling enzymes. This proposal has three specific aims. In the first aim we propose a biochemical approach to investigate the role o f histone H3 and H1 phosphorylation on chromatin structure.
This aim i s addressed by nuclease probing of mononucleosome, the folding of model nucleosomal arrays, and in the recruitment and activity of the SW/SNF family of ATP-dependent chromatin remodeling enzymes. This proposal has three specific aims. In the first aim we propose a biochemical approach to investigate the role of histone H3 and H1 phosphorylation on chromatin structure.
This aim i s addressed by biophysical properties of nucleosomal arrays, and analysis of the accessibility of DNA in the context of a mononucleosome or nucleosomal arrays to restriction enzymes and transcription factors. The objective of aim 2 is to test the hypothesis that phosphorylation of histones H3 and H1 enhances the functioning of SWI/SNF-like chromatin remodeling enzymes. In this aim we propose mononucleosome and nucleosomal array assays to quantitative the remodeling activities of the yeast SWI/SNF, yeast RSC, and human SWI/SNF chromatin remodeling complexes.
This aim will also use novel mammalian cell lines that inducibly express dominant negative SWI/SNF subunits to investigate the role of SWI/SNF in vivo.
Aim 3 3 will investigate the cellular localization of phosphorylated histones H3 and H1, and the localization of human SWI/SNF proteins in mammalians cells.
This aim will be addressed by immunofluorescence of intact cells and nuclear matrix preparations; chromatin immunoprecipitation analyses will also be employed.
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