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， 机械与这种结构竞争。我们的总体策略是 结合生物化学和遗传学的方法来剖析 组蛋白结构域和非组蛋白蛋白质的染色质结构， 功能对酿酒酵母的遗传研究 涉及HMG 1样蛋白SIN 1和HMG 1样蛋白的结构域的鉴定。 组蛋白八聚体，由来自组蛋白H3和H4的残基组成， 在染色质介导的转录抑制中起关键作用， vivo.此外，高等真核生物系统的生化研究和 酵母的遗传分析表明，组蛋白N-末端 在核小体模板上调节转录因子功能。我们 工作假设是，SIN 1，组蛋白八聚体的一个结构域， 组蛋白N-末端调节转录因子与核小体结合 或者通过影响核小体阵列的致密化， 核小体核心内DNA的可及性。该提案利用了 强大的遗传和生物化学的机会， 直接检验这个假设。 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。最 重要的是，我们已经成功地纯化了大量的酵母， 我们有能力在DNA模板上重组的组蛋白。我们 建议用突变的组蛋白重建DNA模板， 在体内定义功能，因此这些研究将提供 独特的机会来解决组蛋白结构域在核小体中的作用 结构和功能。本提案的第一个目的是调查 通过遗传学鉴定的八聚体结构域的功能作用 分析并在染色质介导的转录调控中起关键作用 镇压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 琼脂糖凝胶电泳和分析超离心分析 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假设中起作用的测定： 限制转录因子的可及性。第三个目标将 研究SIN 1蛋白结合核小体阵列的能力 并影响通过定量琼脂糖凝胶分析的阵列压实 电泳和分析超离心。为此，我们将 还使用遗传筛选来鉴定功能上 关于SIN 1第四个目标将定义组蛋白的作用 H4 N-末端结构域在核小体阵列致密化中的作用。的结果 拟议的研究将丰富我们对功能的了解 染色质结构中的非组蛋白和组蛋白结构域， 功能，并提供有价值的见解的机制， 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.