Transcriptional Activation by Reorganizing Chromatin

ATP依赖性染色质重塑蛋白SWI/SNF通过以下途径调节转录和DNA重组： 使DNA更容易被获取本研究的目的是寻找SWI/SNF移动的机制， 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。 最终目标是以时间方式将这两组不同的交互相互关联起来 为核小体重塑过程提供了前所未有的视角。我们建议采用 一种相对较新的技术，扩展了遗传密码，以位点特异性地掺入光反应性氨基， 酸类似物，用于检查体内和体外的蛋白质-蛋白质和蛋白质-DNA相互作用。域 SWI/SNF的催化亚基而不是ATP酶结构域将被系统地研究， 前提是这些结构域对协调调动核小体做出了关键贡献， 与ATP酶结构域合作。已经确定了几个必要的领域， SWI/SNF重塑。单分子磁镊和DNA解旋光阱型实验 将完成，以确定这些结构域是否是重要的DNA易位和核小体重塑。 将检查组蛋白H3乙酰化对SWI/SNF重塑的影响，以确定在哪个阶段， 重塑调节SWI/SNF和RSC的活性。 SWI/SNF具有许多重要的调节作用，例如在干细胞自我更新，细胞分化， 染色质维持和稳定以及肿瘤发生。虽然酵母系统为我们提供了重要的 关于这种酶的功能和相应的模型，仍然存在许多问题， SWI/SNF如何调节染色质结构。在这个建议中，我们继续利用酵母 在生物物理学、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.