Yeast Chromatin Structure and Function

我们研究的总体目标是确定染色体结构如何影响核过程，我们的一般策略之一是识别和表征促进酵母核小体阵列折叠成具有独特生物功能的特殊结构的因素。我们最近的研究表明，在有丝分裂过程中，HMG1样蛋白Sin1p和由组蛋白H3和H4残基组成的组蛋白八聚体的SIN结构域在染色质介导的转录抑制中发挥关键作用。我们的工作假设是，Sin1p与染色质的相互作用需要一个完整的八聚体sin结构域，并且Sin1p通过影响核小体阵列的紧凑来调节有丝分裂转录。我们还认为，酵母接头H1同源物Hhno1p和组蛋白变体Cse4p和Htz1p创建了核小体阵列的特殊折叠结构域，有助于调节基因表达、DNA修复、重组或染色体分离。在接下来的预算期间，我们将继续利用酵母中可用的力量、遗传和生化机会来直接测试这些假设。第一个目的是研究Sin1p在有丝分裂中的作用，并检验Sin1p与组蛋白八聚体SIN结构域相互作用并影响核小体阵列折叠的假设。这些研究将通过间接免疫荧光、染色质结合和染色质免疫沉淀分析来监测作为细胞周期进展的函数的Sin1p与染色质在体内的相互作用。我们还将使用分析超速离心法来表征SIN1P与野生型和SIN-组蛋白八聚体重组的核小体阵列的结合。第二个目标将使用候选基因方法和遗传筛选来识别编码有丝分裂染色质关键成分的基因。第三个目的是研究酵母组蛋白H1(Hho1p)在染色质结构和功能中的作用。在这个目的中，我们将使用染色质免疫沉淀来测试Hho1p是否只针对几个基因座，或者Hho1p是否均匀地分布在酵母染色质中。这个目标将使用分析离心法来研究Hho1p浓缩与酵母组蛋白八聚体重组的核小体阵列的能力。第四个目标将使用分析超速离心法来分析含有Cse4p或Htz1p的核小体阵列的折叠动力学。这些研究将检验这样一种假设，即这些组蛋白变体会产生特殊的染色质结构。

The overall objective of our research is to determine how chromosome structure influences nuclear processes, and one of our general strategies is to identify and characterize the factors that facilitate the folding of yeast nucleosomal arrays into specialized structures with unique biological functions. Our recent studies suggest than an HMG1-like protein, Sin1p, and the SIN domain of the histone octamer comprised of residues from both histones H3 and H4, play crucial roles in chromatin- mediated transcriptional repression during mitosis. Our working hypothesis is that interactions of Sin1p with chromatin requires an intact octamer SIN domain and that Sin1p regulates mitotic transcription by influencing compaction of nucleosomal arrays. We also propose that the yeast linker H1 homologue, Hhno1p, and the histone variants, Cse4p and Htz1p, create specialized, folded domains of nucleosomal arrays that contribute to the regulation of gene expression, DNA repair, recombination, or chromosome segregation. Over the next budget period we will continue to exploit the power genetic and biochemical opportunities available in yeast to directly test these hypotheses. The first aim will investigate the role of Sin1p in mitosis and will test the hypothesis that Sin1p interacts with the histone octamer SIN domain and influences nucleosomal array folding. These studies will be addressed by indirect immunofluorescence, chromatin association, and chromatin immunoprecipitation assays to monitor in vivo interactions of Sin1p with chromatin as a function of cell cycle progression. We will also use analytical ultracentrifugation to characterize the binding of SIN1P to nucleosomal arrays reconstituted with wildtype and sin-histone octamers. The second objective will use both a candidate gene approach and genetic screens to identify genes that encode key components of mitotic chromatin. The third aim will investigate the role of yeast histone H1 (Hho1p) in chromatin structure and function. In this aim we will use chromatin immunoprecipitation to test whether Hho1p is targeted to only a few loci or if Hho1p is uniformly distributed through yeast chromatin. This aim will use analytical centrifugation to investigate the ability of Hho1p to condense nucleosomal arrays reconstituted with yeast histone octamers. The fourth objective will use analytical ultracentrifugation to analyze the folding dynamics of nucleosomal arrays that contain Cse4p- or Htz1p-containing nucleosomes. These studies will test the hypothesis that these histone variants create specialized chromatin structures.