ORGANIZATION OF CHROMATIN BY GLOBAL REGULATORS IN YEAST

将DNA包装到染色质中被广泛认为是真核细胞基因调控的关键步骤。然而，关于转录活性与抑制染色质状态的结构，或这两种状态可能互换的机制，人们知之甚少。这项研究的长期目标是了解染色质是如何组织成功能域的。本提案中的实验将通过分析酵母中的模型调节因子，Tup1p抑制因子来解决这个问题。在上一个资助期完成的实验使我们提出了Tup1p组织染色质抑制转录的假设。我们已经证明Tup1p在体外与组蛋白H3和H4的氨基末端尾部结构域特异性相互作用，并且这些尾部结构域的突变在体内协同破坏抑制。初步数据表明，H3和H4的翻译后乙酰化抑制了Tup1p与这些组蛋白的结合，表明这种修饰可能调节了Tup1p的功能。与此一致的是，受Tup1p抑制的基因在抑制条件下与体内这些组蛋白的乙酰化形式相关联，而不是在激活条件下。此外，在体内引起高乙酰化的特定组蛋白去乙酰化酶活性突变会损害多个靶基因对Tup1p的抑制。最近的数据也表明H3的磷酸化可能会影响Tup1p的功能。H3氨基末端区域（丝氨酸10）保守磷酸化位点的突变严重限制了抑制作用。总之，这些数据提示了tup1p介导的抑制可能被调节的机制，并且更广泛地表明组蛋白修饰的一个重要作用可能是调节调节因子与染色质的关联。这种影响目前尚未得到充分研究，但可能对功能性染色质结构域的组织至关重要。本课题的实验将进一步验证我们的假设，有两个具体目的：1)解剖Tup1p与染色质相互作用的分子性质和功能后果；2)确定特定组蛋白修饰对Tup1p功能的影响，以及在体内如何在Tup1p调控的启动子上建立不同的修饰状态。了解像Tup1p这样的调节蛋白是如何影响染色质结构的，以及这些功能本身是如何被调节的，对于理解正常细胞中基因表达是如何被编程的，以及这些功能的变化是如何导致与疾病状态相关的异常转录模式至关重要。

The packaging of DNA into chromatin is now widely recognized as a key step in gene regulation in eukaryotic cells. However, relatively little is known about the structure of transcriptionally active vs. repressed chromatin states, or the mechanisms by which these two states might be interchanged. The long term goal of this research is to understand how chromatin is organized into functional domains. Experiments in this proposal will address this question through an analysis of a model regulator in yeast, the Tup1p repressor. Experiments completed in the last funding period have led us to the hypothesis that Tup1p organizes chromatin to repress transcription. We have shown that Tup1p interacts specifically with the amino terminal tail domains of histones H3 and H4 in vitro, and that mutations in these tails domains synergistically compromise repression in vivo. Preliminary data indicate that post-translational acetylation of H3 and H4 inhibits Tup1p binding to these histones, suggesting that this modification may modulate Tup1p functions. Consistent with this, genes subject to Tup1p repression are associated with less acetylated forms of these histones in vivo under conditions of repression than under conditions of activation. Moreover, mutations in specific histone deacetylase activities that cause hyperacetylation in vivo compromise Tup1p repression of multiple target genes. Recent data also indicate that phosphorylation of H3 may affect Tup1p functions. Mutation of a conserved phosphorylation site in the amino terminal region of H3 (serine 10) severely limits repression. Together, these data suggest a mechanism by which Tup1p-mediated repression might be modulated, and more globally, indicate that one important role of histone modifications may be to regulate the association of regulatory factors with chromatin. Such affects are understudied at present, but may be centrally important to the organization of functional chromatin domains. Experiments in this proposal will further test our hypothesis with two specific aims: 1) to dissect the molecular nature and functional consequences of Tup1p interactions with chromatin and 2) to determine the effects of specific histone modifications on Tup1p functions, as well as how different modification states are established at Tup1p regulated promoters in vivo. Understanding how regulatory proteins like Tup1p influence chromatin structure, and how such functions are themselves modulated, is crucial to understanding how gene expression is programmed in normal cells, and how changes in these functions lead to abnormal transcription patterns associated with disease states.