Molecular Mechanisms of Global Represion in Yeast

描述(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现象，包括发育和人类疾病，是必要的基础科学知识。这一建议解决了两个具有广泛意义的相关问题：进化上相关的酵母Cyc8-Tup1和人类TLE辅阻遏子转录抑制的分子机制，以及组蛋白修饰的全球模式与转录和维持表观遗传状态之间的关系。为此，我们将结合染色质免疫沉淀、遗传、基因组和生化方法来实施以下项目。首先，我们将讨论Cyc8-Tup1如何介导抑制的三个一般模型的机制和相对重要性：与与RNA聚合酶II相关的中介复合体的相互作用；对染色质的直接影响；空间位阻。我们将确定Cyc8-Tup1抑制的机制步骤(S)，分析Cyc8-Tup1是否足以招募介体并与激活者合作或抑制其招募介体，确定介体复合体中Tup1抑制域的直接靶点，并解决Cyc8-Tup1是否招募组蛋白脱乙酰酶以及它如何介导染色质的长期影响。其次，我们将测试Eaf3通过Eaf3染色域与编码区甲基化的组蛋白之间的相互作用来控制酵母细胞中组蛋白乙酰化的全球模式的模型，该编码区导致Rpd3复合体优先脱乙酰化。我们还将测试这样一种想法，即整个酵母基因组中未被认可的转录水平解释了明显的悖论，即Pall转录延长复合体是组蛋白H3在赖氨酸4和79的全基因组甲基化所必需的。第三，我们将测试在S相期间组蛋白沉积是通过涉及H3-H4四聚体的传统模型还是涉及H3-H4二聚体的新提出的模型发生的，这一问题对如何通过细胞分裂维持表观遗传状态具有重大意义。此外，我们将直接探讨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，作为我们与Affymetrix的Tom Gingera继续合作的一部分，涉及平铺微阵列，我们将以公正的方式在全基因组规模上定义人类TLE蛋白在经历维甲酸诱导分化的HL60细胞中的生理靶点。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联系，这些图谱将在本提案未涵盖的工作中对相同的样本进行。我们将使用RNAi来识别受各种TLE蛋白影响的基因，并将利用功能良好的B-干扰素启动子的衍生物来研究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.