YEAST CHROMATIN STRUCTURE/FUNCTION

拟议研究的总体目标是确定如何 染色体结构影响基因表达和转录 机械与这种结构相抗衡。我们的总体策略是使用 生物化学和遗传学相结合的方法来剖析这些贡献 组蛋白结构域和非组蛋白结构与染色质结构的关系 功能。对酿酒酵母的遗传研究引领了 对HMG1样蛋白SIN1和HMG1的一个结构域的鉴定 组蛋白八聚体，由组蛋白H3和H4残基组成， 在染色质介导的转录抑制中起关键作用 活着。此外，高等真核生物系统中的生化研究和 酵母中的遗传分析表明组蛋白N末端的作用 在核小体模板上调节转录因子的功能。我们的 工作假说是组蛋白八聚体的结构域SIN1和 组蛋白N末端调节转录因子与核小体的结合 要么通过影响核小体阵列的紧凑，要么通过 核小体核心内DNA的可及性。这项建议利用了 酵母中存在强大的遗传和生化机会 直接检验这一假设。 在过去的一年里，我的实验室在开发 拟议研究所需的生化和遗传工具。多数 重要的是，我们已经成功地纯化了大量的酵母 我们有能力将组蛋白重组到DNA模板上。我们 建议用突变的组蛋白重建DNA模板 在体内定义的功能，因此这些研究将提供 解决组蛋白结构域在核小体中的作用的独特机会 结构和功能。这项提案的第一个目的是调查 八聚体结构域的功能作用由基因识别 分析并在染色质介导的转录中发挥关键作用 压抑。这一目标是通过核酸酶消化研究、分析 组蛋白-组蛋白相互作用的盐稳定性，以及定量 琼脂糖凝胶电泳法和分析超速离心法 核小体阵列。第二个目标使用各种足迹 测试该八聚体结构域在以下方面的作用 限制转录因子的可及性。第三个目标将是 研究SIN1蛋白与核小体阵列结合的能力 并影响定量琼脂糖凝胶测定的阵列紧密性 电泳法和分析超速离心法。为了实现这一目标，我们将 也可以使用基因筛查来识别具有功能的蛋白质 与SIN1相关。第四个目标将定义组蛋白的作用 核小体阵列致密中的H4N-末端结构域。结果来自于 建议的研究将会丰富我们对这些功能的认识 染色质结构中的非组蛋白和组蛋白结构域 功能，并提供了对以下机制的宝贵见解 转录受核小体模板的调控。

The overall objective of the proposed research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to use a combined biochemical and genetic approach to dissect the contributions of histone domains and nonhistone proteins to chromatin structure and function. Genetic studies in the yeast Saccharomyces cerevisiae have led to the identification of a HMG1-like protein, SIN1, and a domain of the histone octamer, comprised of residues from both histones H3 and H4, that play crucial roles in chromatin-mediated repression of transcription in vivo. In addition, biochemical studies in higher eukaryotic systems and genetic analyses in yeast have indicated a role for the histone N-termini in modulating transcription factor function on nucleosomal templates. Our working hypothesis is that SIN1, a domain of the histone octamer, and the histone N-termini regulate transcription factor binding to nucleosomes either by influencing compaction of nucleosomal arrays or the accessibility of DNA within nucleosome cores. This proposal exploits the powerful genetic and biochemical opportunities available in yeast to directly test this hypothesis. During the past year, my lab has made significant progress in developing the biochemical and genetic tools required for the proposed studies. Most importantly, we have succeeded in purifying large quantities of yeast histones that we competent for reconstitution onto DNA templates. We propose to reconstitute DNA templates with mutant histones that have been defined functionally in vivo, and thus these studies will provide an unique opportunity to address the role of histone domains in nucleosome structure and function. The first aim of this proposal will investigate the functional role of an octamer domain that was identified by genetic analyses and plays a crucial role in chromatin-mediated transcriptional repression. This aim is addressed by nuclease digestion studies, analysis of the salt stability of histone-histone interactions, and quantitative agarose gel electrophoresis and analytical ultracentrifugation analyses of nucleosome arrays. The second objective uses various footprinting assays to test the hypothesis that this octamer domain plays a role in restricting transcription factor accessibility. The third aim will investigate the ability of the SIN1 protein to bind to nucleosomal arrays and to influence array compaction as assayed by quantitative agarose gel electrophoresis and analytical ultracentrifugation. In this aim we will also use a genetic screen to identify proteins that are functionally related to SIN1. The fourth objective will define the role of the histone H4 N-terminal domain in compaction of nucleosome arrays. The results from the proposed studies will enrich our knowledge of the functions of nonhistone proteins and histone domains in chromatin structure and function as well as provide valuable insights into mechanisms by which transcription is regulated on nucleosomal templates.