Eukaryotic Chromatin Structure and Gene Regulation

我们小组继续研究染色质结构和真核基因表达的调控。在过去的一年里，我们已经取得了重大进展，了解ATP依赖的染色质重塑的机制，果蝇Nucleosome Remodeling Factor（NF）。先前，我们证明了BNF通过诱导核小体“滑动”来重塑染色质，核小体“滑动”是组蛋白八聚体的相对运动，而不会从DNA中发生不可挽回的位移。我们现在已经成功地重建了四个亚基的重组蛋白质，从而允许亚基的要求和蛋白质结构域的突变研究的分析。我们已经发现，两个最大的BNF组分，BNF 301和ISWI ATP酶，足以重建有效和准确的核小体滑动活性。ISWI是核小体滑动的引擎，而ISWI F301增强了核小体移动的效率和位置特异性。为了实现这一点，BNF 301与核小体进行了几次不同的接触。我们已经定义了一个HMGA（HMGI/Y）样的DNA弯曲结构域在ADAP 301和证明其功能的重要性的滑动机制。BMPF 301还显示与序列特异性转录因子的相互作用，从而为BMPF复合物靶向募集到特定基因提供了基础。我们的位置，以阐明剩余的功能结构域的ISWIF 301，并探讨如何耦合的ISWIF 301的ATP酶导致核小体运动的染色质。我们已经分离出了果蝇ADF 301基因的突变，并研究了它的表型。我们还在更大的12组分染色质重塑复合物（INO 80复合物）的研究方面取得了很大进展。我们已经完成了所有12个亚基的胰蛋白酶肽的质谱鉴定，并正在进行遗传和生化实验，以阐明这种新的染色质重塑复合物的机制和生理功能。我们的研究继续为染色质和转录之间的重要联系提供新的见解。

Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression. In the past year we have made significant progress towards understanding the mechanism of ATP-dependent chromatin remodeling by Drosophila NURF (Nucleosome Remodeling Factor). Previously, we demonstrated that NURF remodels chromatin by inducing nucleosome "sliding," the relative movement of a histone octamer without irretrievable displacement from DNA. We have now successfully reconstituted the four-subunit NURF from recombinant proteins, thus allowing analysis of subunit requirements and a mutational study of protein domains. We have found that the two largest NURF components, NURF301 and the ISWI ATPase, are sufficient to reconstitute efficient and accurate nucleosome sliding activity. ISWI is the engine of nucleosome sliding, while NURF301 enhances efficiency and positional specificity of nucleosome movement. To achieve this, NURF301 makes several distinct contacts with nucleosomes. We have defined a HMGA (HMGI/Y)-like DNA-bending domain in NURF301 and demonstrated its functional importance to the sliding mechanism. NURF301 also shows interactions with sequence-specific transcription factors, thus providing a basis for targeted recruitment of the NURF complex to specific genes. We are in position to elucidate the remaining functional domains of NURF301, and to investigate how coupling of NURF301 to the ISWI ATPase leads to nucleosome movements in chromatin. We have isolated mutations for the Drosophila NURF301 gene, and have studied its phenotypes. We are also making excellent progress on studies of a much larger, 12-component chromatin remodeling complex (the INO80 complex). We have completed identification of all 12 subunits by mass spectrometry of tryptic peptides, and are conducting genetic and biochemical experiments to elucidate the mechanism and physiological functions of this new chromatin remodeling complex. Our studies continue to provide new insights to the important link between chromatin and transcription.