Molecular Mechanisms of Global Represion in Yeast

描述（由申请人提供）：真核生物调节基因表达和染色质结构的分子机制对于理解许多复杂的生物学现象（包括发育和人类疾病）所必需的基础科学知识非常重要。这一建议解决了两个相关的问题，具有广泛的意义：转录抑制的分子机制的进化相关的酵母Cyc 8-Tup 1和人类TLE辅阻遏物，和组蛋白修饰转录和维持表观遗传状态的全球模式之间的关系。为此，我们将结合联合收割机染色质免疫沉淀，遗传学，基因组学和生物化学的方法来进行以下项目。首先，我们将讨论Cyc 8-Tup 1如何介导阻遏的三种一般模型的机制和相对重要性：与RNA聚合酶II相关的介体复合物的相互作用;对染色质的直接影响;空间干扰。我们将确定Cyc 8-Tup 1抑制的机制步骤，分析Cyc 8-Tup 1是否足以招募Mediator并与激活剂合作或抑制激活剂招募Mediator，确定Mediator复合物内Tup 1抑制结构域的直接靶点，并解决Cyc 8-Tup 1是否招募组蛋白脱乙酰酶及其如何介导对染色质的长期影响。第二，我们将测试的模型，Eaf 3控制组蛋白乙酰化在酵母细胞中的全球模式，通过Eaf 3的染色体结构域和甲基化的组蛋白之间的相互作用，在编码区，导致优先脱乙酰化的Rpd 3复合物。我们还将测试的想法，整个酵母基因组的转录未被赞赏的水平占明显的悖论，即Pall转录延伸复合物是所需的组蛋白H3在赖氨酸4和79的全基因组甲基化。第三，我们将测试组蛋白沉积是否发生在S期的传统模型涉及H3-H4四聚体或一个新提出的模型涉及H3-H4二聚体，一个问题与表观遗传状态是如何通过细胞分裂维持的重大影响。此外，我们将直接解决转录活性和组蛋白解离之间的关系和机制，并测量转录相关（即非复制）核小体沉积的速率。第四，作为我们继续与Affyeland的Tom Gingeras合作的一部分，涉及平铺微阵列，我们将以公正的方式和全基因组规模定义HL 60细胞中人TLE蛋白的生理靶点，这些细胞经历维甲酸诱导的分化。这些结果将与RNA转录本、组蛋白修饰、染色质修饰活性和转录调控因子的详细图谱相关联，这些图谱将在本提案未涵盖的工作中对相同样品进行。我们将使用RNAi来鉴定受各种TLE蛋白影响的基因，并将通过使用充分表征的B-干扰素启动子的衍生物来解决TLE蛋白的抑制机制。

DESCRIPTION (provided by applicant): The molecular mechanisms by which eukaryotes regulate gene expression and chromatin structure are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including development and human disease. This proposal addresses two related issues of broad significance: the molecular mechanisms of transcriptional repression by the evolutionarily related yeast Cyc8-Tup1 and human TLE corepressors, and the relationship between global patterns of histone modifications to transcription and maintenance of epigenetic states. To do this, we will combine chromatin immunoprecipitation, genetic, genomic, and biochemical approaches to carry out the following projects. First, we will address the mechanisms and relative importance of three general models for how Cyc8-Tup1 mediates repression: interaction with the Mediator complex that associates with RNA polymerase II; direct effects on chromatin; steric interference. We will determine the mechanistic step(s) inhibited by Cyc8-Tup1, analyze whether Cyc8-Tup1 is sufficient to recruit Mediator and cooperates with or inhibits activators from recruiting Mediator, identify direct targets of the Tup1 repression domain within the Mediator complex, and address whether Cyc8-Tup1 recruits histone deacetylases and how it mediates long-range effects on chromatin. Second, we will test the model that Eaf3 controls the global pattern of histone acetylation in yeast cells via an interaction between Eaf3 chromodomains and methylated histones in the coding region that causes preferential deacetylation by the Rpd3 complex. We will also test the idea that an unappreciated level of transcription throughout the yeast genome accounts for the apparent paradox that the Pall transcriptional elongation complex is required for genome-wide methylation of histone H3 at lysines 4 and 79. Third, we will test whether histone deposition during S phase occurs by the conventional model involving H3-H4 tetramers or a newly proposed model involving H3-H4 dimers, an issue with major implications for how epigenetic states are maintained through cell division. In addition, we will directly address the relationship and mechanism between transcriptional activity and histone dissociation, and measure the rate of transcription-associated (i.e. non-replicative) nucleosome deposition. Fourth, as part of our continuing collaboration with Tom Gingeras at Affymetrix that involves tiled microarrays, we will define, in an unbiased manner and on a whole-genome scale, the physiological targets of human TLE proteins in HL60 cells undergoing retinoic acid-induced differentiation. These results will be linked to detailed maps of RNA transcripts, histone modifications, chromatin-modifying activities, and transcriptional regulatory factors that will be performed on the identical samples in work not covered by this proposal. We will use RNAi to identify genes affected by the various TLE proteins, and will address the mechanism of repression by TLE proteins by using derivatives of the well characterized B-interferon promoter.