TRANSCRIPTIONAL ACTIVATION BY REORGANIZING CHROMATIN

Southern Illinois University Carbondale, Carbondale IL

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Abstract

DESCRIPTION (Adapted from application): An understanding of the mechanism of eukaryotic gene regulation is indispensable in trying to cure cancer. The difference between a normal cell and a transformed or malignant cell is largely due to alterations in the transcription of specific genes. Recently it has become apparent that one very important aspect of gene regulation is the manner in which the DNA is packaged into chromatin & how the chromatin can be remodeled so that DNA is accessible to the transcriptional machinery. Current studies have demonstrated the existence of large protein complexes that remodel chromatin with the subsequent effect of gene activation. The structures of these protein complexes and their mechanism of chromatin remodeling are not known and in only a few cases have the proteins involved been identified. The work in this application proposes to use positioned nucleosomes, protein-protein crosslinking, and site-directed DNA-protein photocrosslinking to study the structure of the yeast SWI/SNF complexes and the underlying mechanism whereby they remodel chromatin in an ATP-dependent fashion. Data clearly indicate that SWI/SNF plays a pivotal role in gene expression. First is the extensive literature showing that SWI/SNF acts as a transcriptional activator. Second, biochemical and genetic data demonstrate that SWI/SNF functions as a chromatin remodeling machine. And third, SWI/SNF appears to be important enough that it may be carried around with the Pol II holoenzyme in approximately stoichiometric amounts. Finally, there is even evidence that links some cancers with defective SWI/SNF complexes or to altered interactions with SWI/SNF. The questions addressed by this proposal are the following. First, what happens to the histones as the nucleosome is remodeled by different chromatin remodeling factors such as the yeast SWI/SNF and RSC complexes? Are the nucleosomes displaced from DNA, or is the structure of the nucleosome altered to make the DNA more accessible and if so, how is it altered? Second, what are the mechanistic steps involved in the remodeling of chromatin? What is the basis of the ATP-driven chromatin remodeling activity of SWI/SNF? How does the chromatin remodeling factor contact the nucleosomal DNA and alter/disrupt the contacts of hstones with DNA? What are the structural and functional differences of the different chromatin remodeling factors present in yeast? Third, how does a chromatin remodeling factor get recruited to a particular site on DNA? Are there direct interactions between SWI/SNF complexes and transcriptional activators, or are there adaptor factors that are required for the selective association of the chromatin remodeling factor?