Regulation of chromatin structure and gene expression

本研究的长期目标是从分子水平了解表观遗传过程 基因沉默和染色质重塑。果蝇中的PEV已经成为研究PEV的主要范例。 表观遗传调控进化保守决定因子的鉴定和遗传分析 染色质结构和基因沉默。我们提出的证据表明，JIL-1组蛋白H3 S10染色体 激酶通过拮抗异染色质化和基因沉默来维持常染色质区域 通过甲基转移酶介导的组蛋白H3 K9二甲基化和HP 1募集在异位位置。 因此，为了了解果蝇异染色质形成和基因沉默的调控， 将是至关重要的，以确定分子机制的JIL-1的作用，在这一过程中和JIL-T的位置 在遗传层次中。因此，我们将检验JIL-1的功能是平衡利差的假设 异染色质因素的1）确定染色质标记物分布的变化， 诊断JIL-1突变背景中的活性（常染色质）或沉默（异染色质）染色质， 通过2）进行遗传实验以确定JIL-1发挥功能以维持 染色体结构域并确定JIL-Ts在遗传层次中的位置，以及通过3）确定 JIL-1活性丧失和染色质结构紊乱的分子机制 变种人背景此外，我们提出，在JIL-1功能丧失中观察到的PEV变化 突变体将伴随着染色质中核小体水平的染色质结构的变化 context-dependent方式。为了验证这些假设，我们将进行一系列的遗传和生物化学研究。 实验旨在研究JIL-1如何影响核小体组织和PEV使用染色体 倒位和将报告基因插入不同的染色质环境。我们将直接测试 染色质结构改变是由组蛋白磷酸化状态引起的假说 通过将JIL-1靶向至异位染色体位置的H3 S10。最后，我们将定义 JIL-1是PEV染色质结构调节所必需的，并决定激酶活性的作用 在该过程中，通过产生“激酶死亡”构建体并表达JIL-1的突变构建体， 在空突变果蝇中转基因。基因沉默是一个重要的发育过程， 包括癌症在内的人类健康问题。因此，JIL-1的拟议研究将提供重要的新信息， 深入了解激酶活性如何调节染色质结构和基因的分子机制 与人类直接相关的法规。

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 inJIL-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.