Eukaryotic Chromatin Structure and Gene Regulation

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Our group has continued studies of chromatin structure and regulation of gene expression. In the past year we have used the RNAi technique to validate candidates from two-hybrid screens for regulators of the Drosophila Heat Shock Transcription Factor (HSF). We found that the molecular chaperones DROJ1 and HSP70 cooperate to repress the activity of HSF. Depletion of both chaperones from SL2 cells leads to full induction of HSF activity under nonstress conditions. These findings provide strong support for the feedback inhibition model for heat shock regulation. We have also completed the expression and purification of all four subunits of the Drosophila Nucleosome Remodeling Factor (NURF) in a baculovirus expression system, and found that the recombinant proteins associate to form a complex with specific activity equal to native NURF. We are continuing biochemical and genetic studies of the NURF subunits to elucidate the mechanism and physiological functions of ATP-dependent nucleosome mobility. To study other chromatin remodeling proteins, we purified a new chromatin remodeling complex from Saccharomyces cerevisiae (the INO80 complex) composed of ~12 components that is implicated in transcription and DNA damage repair.