Transcriptional Activation by Reorganizing Chromatin

依赖于ATP的染色质重构体SWI/SNF通过以下方式调节转录和DNA重组 让DNA更容易获取。本研究的目的是找出SWI/SNF使用的运动机制 并分解核小体。核小体重塑将在两个水平上进行检查：组蛋白的变化- DNA相互作用以及SWI/SNF与组蛋白和DNA相互作用的相应变化。 最终的目标是以时间的方式将这两组不同的交互相互关联 为核小体重塑过程提供一个前所未有的视角。我们建议将 相对较新的将遗传密码扩展到位点的技术--特定地结合了一种光反应氨基 酸性类似物，用于检测体内和体外的蛋白质-蛋白质和蛋白质-DNA相互作用。中的域 除了ATPase结构域之外，SWI/SNF的催化亚基将被系统地研究 前提是这些结构域对协调和动员核小体做出关键贡献 与ATPase域名合作。已经确定了几个域，这些域是 SWI/SNF重塑。单分子磁钳和DNA解卷光陷式实验 以确定这些结构域是否对DNA易位和核小体重塑重要。 组蛋白H3乙酰化对SWI/SNF重塑的影响将被检测，以确定在哪个阶段 重塑它可调节SWI/SNF和RSC的活性。 SWI/SNF在干细胞自我更新、细胞分化、 染色质的维持和稳定，以及肿瘤的发生。虽然酵母系统为我们提供了重要的 关于这种酶是如何发挥作用的以及相应的模型，仍然存在许多问题，如 SWI/SNF是如何调节染色质结构的。在这项提案中，我们继续利用酵母 在生物物理、生物化学和分子遗传学水平上研究这些问题的系统。

The ATP-dependent chromatin remodeler SWI/SNF regulates transcription and DNA recombination by making DNA more accessible. The objective of this study is to find the mechanism used by SWI/SNF to move and disassemble nucleosomes. Nucleosome remodeling will be examined at two levels: changes in histone- DNA interactions and the corresponding changes in the interactions of SWI/SNF with histones and DNA. Ultimately the goal is to correlate these two different sets of interactions with each other in a temporal manner to provide an unprecedented view of the process of nucleosome remodeling. We propose to apply the relatively new technology of an expanded genetic code to site-specifically incorporate a photoreactive amino acid analog for examining protein-protein and protein-DNA interactions both in vivo and in vitro. Domains in the catalytic subunit of SWI/SNF other than the ATPase domain will be systematically studied with the basic premise that these domains make critical contributions to mobilizing nucleosomes in coordination and cooperation with the ATPase domain. Several domains have already been identified that are necessary for SWI/SNF remodeling. Single molecule magnetic tweezer and DNA unwinding optical trap type experiments will be done to determine if these domains are important for DNA translocation and nucleosome remodeling. The effect of histone H3 acetylation on SWI/SNF remodeling will be examined to determine at which stage in remodeling it modulates the activity of SWI/SNF and RSC. SWI/SNF has many important regulatory roles such as in stem cell self renewal, cellular differentiation, chromatin maintenance and stability, and oncogenesis. While the yeast system has provided us with important insights as to how this enzyme functions and the corresponding models, there still remain many questions as to how SWI/SNF regulates chromatin structure. In this proposal we continue to take advantage of the yeast system to examine these questions at a biophysical, biochemical, and molecular genetic level.