Organization of Chromatin by Global Regulators in Yeast

描述（由申请人提供）：染色质重塑现在被认为是基因调控的关键步骤。我们研究的长期目标是了解染色质结构的变化是如何产生的，以及这些变化如何增强或抑制转录。在这个建议中的实验将解决这些基本问题，通过分析的分子机制的模型辅阻遏复合物介导的酵母，Tup 1-Ssn 6的镇压。我们以前证明，Tup 1直接与乙酰化不足的组蛋白H3和H4亚型相互作用。我们还表明，Tup 1-Ssn 6介导的阻遏被废除后，同时破坏3 HDAC基因在酵母中，RPD 3，HOS 1和HOS 2。组蛋白中的突变削弱了与Tup 1在体外的相互作用，通过使靶启动子处的Tup 1相互作用不稳定而损害了体内的抑制。这些发现使我们建立了一个模型，其中Tup 1-Ssn 6复合物通过序列特异性阻遏物被募集到靶基因，反过来，募集一种或多种组蛋白修饰活性，以产生一种自我增强的抑制性染色质状态。然而，我们不知道辅阻遏物是否使用相同的工具，并在不同类型的靶基因上建立相同的结构。我们也不知道阻遏机制中事件的顺序，或者其他类型的染色质重塑活动是否也有助于阻遏。我们最近发现，Ssn 6除了与Tup 1共享的功能外，还可能在基因调控中具有一些单独的功能。然而，我们对这些独立的Ssn 6函数的信息很少。为了解决这些问题，我们将1）确定Tup 1-Ssn 6复合物中的HDAC相互作用结构域，并确定辅阻遏物是否同时招募多个HDAC; 2）确定Ssn 6在特定基因的调控中是否独立于Tup 1发挥功能; 3）定义并比较Tup 1-Ssn 6募集、HDAC募集、组蛋白去乙酰化、4）建立体外系统，进一步明确阻遏机制。Tup 1与TLE/groucho和TBL 1/TBLR 1辅阻遏物具有相同的结构和功能特征，这些特征在胚胎发育过程中控制基因表达以及响应高等生物中的特定信号转导途径中非常重要。我们在酵母中的Tup 1-Ssn 6的研究将进一步我们的理解一般辅阻遏物的功能，并将提供新的见解TLE/groucho和TBL家族成员在正常细胞和疾病状态下的功能。

DESCRIPTION (provided by applicant): Chromatin remodeling is now recognized as a key step in gene regulation. The long-term goal of our research is to understand how changes in chromatin structure are brought about, and how these changes enhance or repress transcription. Experiments in this proposal will address these fundamental questions via an analysis of the molecular mechanisms underlying repression mediated by a model corepressor complex in yeast, Tup1-Ssn6. We demonstrated previously that Tup1 interacts directly with underacetylated isoforms of histones H3 and H4. We also showed that Tup1-Ssn6 mediated repression is abrogated upon simultaneous disruption of 3 HDAC genes in yeast, RPD3, HOS1, and HOS2. Mutations in histones that weaken interactions with Tup1 in vitro compromise repression in vivo by destabilizing Tup1 interactions at target promoters. These findings led us to a model wherein Tup1-Ssn6 complexes are recruited to target genes via sequence-specific repressers, and in turn, recruit one or more histone modifying activities to create a self-reinforcing, repressive chromatin state. However, we do not know whether the corepressor uses the same tools and establishes the same kinds of structures at different types of target genes. We also do not know the order of events in the repression mechanism, or whether other types of chromatin remodeling activities also contribute to repression. We recently discovered that Ssn6 might have some separate functions in gene regulation, in addition to functions shared with Tup1. However, we have little information regarding these independent Ssn6 functions. To address these questions, we will 1) Define HDAC interaction domains within the Tup1-Ssn6 complex and determine whether multiple HDACs are recruited simultaneously by the corepressor; 2) Determine whether Ssn6 functions independently of Tup1 in the regulation of specific genes; 3) Define and compare the kinetics of Tup1-Ssn6 recruitment, HDAC recruitment, histone deacetylation, etc. at specific target genes and 4) Establish an in vitro system to further define the mechanism of repression. Tup1 shares structural and functional features with the TLE/groucho and the TBL1/TBLR1 corepressors that are important in controlling gene expression during embryonic development and in response to specific signal transduction pathways in higher organisms. Our studies of Tup1-Ssn6 in yeast will further our understanding of corepressor functions in general and will provide new insights to the functions of TLE/groucho and TBL family members both in normal cells and in disease states.