MOLECULAR ANALYSIS OF ATP DEPENDENT CHROMATIN REMODELING

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The broad, long-term goal of the proposed study is to determine, at a molecular level, the mechanisms of the regulation of chromatin structure. This study is important because the understanding of chromatin regulation is a necessary prerequisite to the elucidation of the mechanisms of many essential biological processes, such as transcription, recombination and DNA repair, all of which are controlled by chromatin structure. Determining the mechanisms of chromatin regulation in turn will lead to a better understanding of the mechanisms underlying diseases such as cancer, which often results from aberrant regulation of gene expression, recombination, or DNA repair. To this end, the in vivo functions and the mechanisms of actions of ATP- dependent chromatin regulators (NURF-like factors, NLFs), which we recently identified in our model organism, Saccharomyces cerevisiae, will be studied. The specific aims of this project are: (1) Purify and identify the subunits of the NLFs. (2) Create null mutants of the NLF subunits and screen for the phenotypes. (3) Carry out DNA chip screens to identify the in vivo targets of the NLFs, as well as a high-copy number suppressor screen and a synthetic-lethal screen to identify genes with the related functions to the NLFs in vivo. (4) Utilize purified NLF complexes and chromatin assembled in vitro to characterize the mechanisms of actions of the NLFs. Our work will involve molecular techniques such as gene cloning, mutagenesis, PCR, biochemical fractionation, in vitro chromatin assembly and analysis, and protein chemistry, as well as yeast molecular genetics.