Role of Set2 and H3 methylation in chromatin function

描述（申请人提供）：真核生物基因表达的调控涉及染色质结构的精确控制。虽然对染色质纤维产生和维持不同功能域的操作知之甚少，但组蛋白翻译后修饰显然是这一过程的核心。该建议的长期目标是阐明组蛋白甲基化在染色质功能中的作用。为了实现这一点，我们将描述最近在酵母中发现的两种独立的组蛋白甲基转移酶（HMT）活性的功能。其中一种活性是Set2，它是酵母中唯一负责赖氨酸36 （K36）上的组蛋白H3甲基化的酶，赖氨酸36是一个从酵母到人类保守的修饰位点。新的证据表明，set2介导的H3 K36甲基化参与葡萄糖抑制，但对这种酶或其甲基化位点知之甚少。鉴于最近对组蛋白甲基化作用的见解，我们假设这些酶调节基因表达，部分是通过向染色质中同源甲基化位点募集下游调节因子。为此，我们将结合生物化学、遗传学和免疫学来确定Set2中负责基因抑制和组蛋白甲基化的结构域，表征Set2的相互作用伙伴，鉴定结合K36甲基化H3的蛋白质，并确定H3 K36甲基化与其他组蛋白修饰一起调节受Set2影响的基因转录的程度。我们还将确定受Set2/H3 K36甲基化影响的其他染色体区域和细胞通路，以努力了解该酶的全局功能。第二种HMT活性尚未确定，但已通过色谱法高度纯化。我们将通过生化方法鉴定这种酶，并进一步表征其甲基化位点，并确定它们在染色质中发生的程度。由于组蛋白修饰在基于染色质的功能中起着至关重要的作用，我们对介导它们的酶及其功能意义的理解的进展可能会对与人类健康和疾病相关的问题产生重大影响。

DESCRIPTION (provided by applicant): Regulation of eukaryotic gene expression involves the precise control of chromatin structure. While the manipulation of the chromatin fiber to create and maintain distinct functional domains is poorly understood, histone post-translational modifications are clearly central to this process. The long-range objective of this proposal is to elucidate roles of histone methylation in chromatin function. To achieve this, we will characterize the functions of two independent histone methyltransferase (HMT) activities recently identified in yeast. One of these activities is Set2, which is the sole enzyme in yeast responsible for methylating histone H3 on lysine 36 (K36), a site of modification conserved from yeast to humans. New evidence suggests that Set2-mediated H3 K36 methylation participates in glucose repression, but little else is known about this enzyme or its methylation site. Given recent insights into the roles of histone methylation, we hypothesize that these enzymes regulate gene expression, in part, through the recruitment of down-stream regulatory factors to their cognate methylation sites in chromatin. To that end, we will use a combination of biochemistry, genetics and immunology to define the domains in Set2 responsible for gene repression and histone methylation, characterize Set2's interacting partners, identify proteins that bind K36-methylated H3 and determine the extent to which H3 K36 methylation functions with other histone modifications to regulate the transcription of a gene affected by Set2. We will also determine other chromosomal regions and cellular pathways affected by Set2/H3 K36 methylation in an effort to understand the global functions of this enzyme. The second HMT activity is not yet identified but has been highly purified by chromatography. We will identify this enzyme by biochemical approaches and further characterize its site(s) of methylation and determine the extent to which they occur in chromatin. Since histone modifications play a vital role in chromatin-based functions, advances in our understanding of the enzymes that mediate them, as well as their functional significance, is likely to have a major impact on issues related to human health and disease.