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学功能的专门结构的因素。我们最近的研究表明，HMG 1样蛋白Sin 1 p和由组蛋白H3和H4残基组成的组蛋白八聚体的SIN结构域在有丝分裂期间染色质介导的转录抑制中起关键作用。我们的工作假设是Sin 1 p与染色质的相互作用需要一个完整的八聚体SIN结构域，Sin 1 p通过影响核小体阵列的压实来调节有丝分裂转录。我们还提出，酵母接头H1同源物，Hhno 1 p，和组蛋白的变体，Cse 4p和Htz 1 p，创建专门的，折叠结构域的核小体阵列，有助于基因表达，DNA修复，重组，或染色体分离的调节。在下一个预算期间，我们将继续利用酵母中的动力遗传和生物化学机会来直接测试这些假设。第一个目标是研究Sin 1 p在有丝分裂中的作用，并检验Sin 1 p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测定来监测Sin 1 p与染色质的体内相互作用作为细胞周期进程的函数。我们还将使用分析性超离心来表征SIN 1 P与用野生型和SIN-组蛋白八聚体重构的核小体阵列的结合。第二个目标将使用候选基因的方法和遗传筛选，以确定编码有丝分裂染色质的关键成分的基因。第三个目标是研究酵母组蛋白H1（Hho 1 p）在染色质结构和功能中的作用。在这个目标中，我们将使用染色质免疫沉淀测试是否Hho 1 p是针对只有几个位点，或者如果Hho 1 p是均匀分布在酵母染色质。这一目标将使用分析离心法来研究Hho 1 p浓缩用酵母组蛋白八聚体重构的核小体阵列的能力。第四个目标将使用分析超离心来分析包含Cse 4p或Htz 1 p的核小体的核小体阵列的折叠动力学。这些研究将检验这些组蛋白变体产生专门的染色质结构的假设。

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.