Eukaryotic Chromatin Structure and Gene Regulation

我们小组继续研究染色质结构和基因表达调控。在过去的一年里，我们使用RNAi技术来验证来自双杂交筛选的果蝇热休克转录因子(HSF)调节因子的候选基因。我们发现分子伴侣DROJ1和HSP70协同抑制HSF的活性。在非应激条件下，SL2细胞中这两个伴侣蛋白的缺失会导致HSF活性的完全诱导。这些发现为热休克调节的反馈抑制模型提供了有力的支持。我们还在杆状病毒表达系统中完成了果蝇核小体重塑因子(NURF)全部四个亚基的表达和纯化，发现重组蛋白相互结合形成了一个具有与天然NURF相同的比活性的复合体。我们正在继续对NURF亚基进行生化和遗传学研究，以阐明依赖于ATP的核小体移动的机制和生理功能。为了研究其他染色质重塑蛋白，我们从酿酒酵母中纯化了一个新的染色质重塑复合体(INO80复合体)，它由~12个组分组成，与转录和DNA损伤修复有关。

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.