Organization of Chromatin by Global Regulators in Yeast

描述（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，以及这些变化是如何增强或抑制转录的。本实验将通过分析酵母中一个模型辅抑制复合物Tup1-Ssn6介导的抑制的分子机制来解决这些基本问题。我们之前证明了Tup1直接与组蛋白H3和H4的低乙酰化亚型相互作用。我们还发现，当酵母、RPD3、HOS1和HOS2中的3个HDAC基因同时被破坏时，Tup1-Ssn6介导的抑制被取消。组蛋白的突变在体外减弱了与Tup1的相互作用，从而破坏了Tup1在目标启动子上的相互作用，从而损害了体内的抑制。这些发现使我们发现了一种模型，其中Tup1-Ssn6复合物通过序列特异性抑制因子被招募到靶基因，并反过来招募一种或多种组蛋白修饰活性来创建自我强化，抑制染色质状态。然而，我们不知道这些共抑制因子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。我们也不知道抑制机制中的事件顺序，或者其他类型的染色质重塑活动是否也有助于抑制。我们最近发现Ssn6除了与Tup1共享的功能外，在基因调控中可能还有一些单独的功能。然而，我们对这些独立的Ssn6功能知之甚少。为了解决这些问题，我们将1)定义Tup1-Ssn6复合体内的HDAC相互作用域，并确定多个HDAC是否同时被辅抑制因子募集；2)确定Ssn6是否独立于Tup1调控特定基因；3)明确并比较特定靶基因上Tup1-Ssn6募集、HDAC募集、组蛋白去乙酰化等过程的动力学；4)建立体外系统，进一步明确抑制机制。Tup1与TLE/groucho和TBL1/TBLR1共抑制因子具有相同的结构和功能特征，这些特征在控制胚胎发育过程中的基因表达和响应高等生物的特定信号转导途径中发挥重要作用。我们对酵母中Tup1-Ssn6的研究将进一步加深我们对协抑制因子功能的总体理解，并将为TLE/groucho和TBL家族成员在正常细胞和疾病状态下的功能提供新的见解。

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.