Regulation of chromatin structure and gene expression

描述（由申请人提供）：本研究的长期目标是获得对基因沉默和染色质重塑的表观遗传过程的分子理解。果蝇中的PEV已成为鉴定和遗传分析染色质结构表观遗传调节和基因沉默的进化保守决定因素的主要范式。我们提出的证据表明，JIL-1组蛋白H3 S10染色体激酶的功能，以维持常染色质区，通过拮抗异染色质化和基因沉默在异位位置由甲基转移酶介导的组蛋白H3 K9二甲基化和HP 1招聘。因此，为了了解果蝇异染色质形成和基因沉默的调控，确定JIL-1在这一过程中的作用的分子机制和JIL-Ts在遗传层次中的位置将是至关重要的。因此，我们将通过以下方式检验JIL-1发挥平衡异染色质因子扩散的功能的假设：1）确定诊断活动性染色体病的染色质标记物分布的变化，（常染色质）或沉默JIL-1突变体背景中的（异染色质）染色质，通过2）进行遗传实验以确定体内途径，其中JIL-1起维持染色体结构域的作用并确定JIL-1的表达。Ts在遗传层次中的位置，以及3）确定JIL-1突变背景中活力丧失和染色质结构扰动的分子机制。此外，我们提出，在JIL-1功能丧失突变体中观察到的PEV变化将伴随着核小体水平上染色质结构的变化，染色质上下文依赖性的方式。为了验证这些假设，我们将进行一系列遗传和生化实验，旨在研究JIL-1如何影响核小体组织和PEV使用染色体倒位和插入到不同的染色质环境的报告基因。我们将通过将JIL-1靶向到异位染色体位置来直接检验染色质结构的改变是由组蛋白H3 S10的磷酸化状态引起的假设。最后，我们将定义特定的结构域的JIL-1的染色质结构的调控是必要的PEV和确定激酶活性在这一过程中的作用，通过产生“激酶死”的构建体和表达突变的构建体的JIL-1转基因无效突变苍蝇。基因沉默是与包括癌症在内的许多人类健康问题相关的关键发育过程。因此，JIL-1的拟议研究将为激酶活性如何调节与人类直接相关的染色质结构和基因调控的分子机制提供重要的新见解。

DESCRIPTION (provided by applicant): The long term objective of this study is to gain a molecular understanding of epigenetic processes of gene silencing and chromatin remodeling. PEV in Drosophila has served as a major paradigm for the identification and genetic analysis of evolutionary conserved determinants of epigenetic regulation of chromatin structure and gene silencing. We present evidence that the JIL-1 histone H3S10 chromosomal kinase functions to maintain euchromatic regions by antagonizing heterochromatization and gene silencing at ectopic locations by methyltransferase mediated histone H3K9 dimethylation and HP1 recruitment. Consequently, to understand regulation of heterochromatin formation and gene silencing in Drosophila it will be crucial to determine the molecular mechanisms of JIL-1's role in this process and JIL-Ts placement in the genetic hierarchy. Thus, we will test the hypothesis that JIL-1 functions to counterbalance the spread of heterochromatic factors by 1) determining the changes in the distribution of chromatin markers that are diagnostic for active (euchromatic) or silenced (heterochromatic) chromatin in JIL-1 mutant backgrounds, by 2) conducting genetic experiments to define the in vivo pathway in which JIL-1 functions to maintain chromosomal domains and determine JIL-Ts place in the genetic hierarchy, and by 3) determining the molecular mechanisms underlying the loss of viability and perturbation of chromatin structure in JIL-1 mutant backgrounds. Furthermore, we propose that changes in PEV observed in JIL-1 loss-of-function mutants will be accompanied by changes in chromatin structure at the nucleosomal level in a chromatin context-dependent manner. To test these hypotheses we will conduct a series of genetic and biochemical experiments designed to examine how JIL-1 affects nucleosome organization and PEV using chromosomal inversions and insertions of a reporter gene into different chromatin environments. We will directly test the hypothesis that alterations in chromatin structure are caused by the phosphorylation state of histone H3S10 by targeting JIL-1 to ectopic chromosome locations. Finally, we will define the specific domains of JIL-1 that are necessary for chromatin structure regulation in PEV and determine the role of kinase activity in this process by generating "kinase dead" constructs and expressing the mutated constructs of JIL-1 transgenically in null mutant flies. Gene silencing is a critical developmental process relevant to many human health problems that include cancer. Thus, the proposed studies of JIL-1 will provide important new insights into the molecular mechanisms of how kinase activity modulates chromatin structure and gene regulation that are directly relevant to humans.