Regulation of chromatin structure and gene expression by H3S10 phosphorylation

描述(由申请人提供)： 项目概述/摘要近年来发现了一些在染色质组织、重塑和表观遗传修饰中起关键作用的分子。然而，我们对哪些是关键的调控因子，或者以特定的表观遗传修饰为标志的染色质结构是如何建立的了解还远远不完整。我们已经提出证据表明，表观遗传组蛋白H3S10在间期被JIL-1激酶磷酸化是通过拮抗果蝇异染色化和基因沉默而调节常染色质区域的关键因素。因此，要了解果蝇异染色质形成和基因沉默的调控，H3S10ph标记在这一过程中发挥作用的分子机制将是至关重要的。基于我们以前的发现，我们提出了一个模型，其中JIL-1激酶活性和组蛋白H3S10的磷酸化通过调节促进基因表达抑制和激活的因子之间的动态平衡来对抗异染色化。这一假说背后的分子机制将在三个具体目标中进行探索：1)在第一个目标中，我们将检验这一假说，即H3S10磷酸化通过使用LacI-Tethering系统异位诱导H3S10磷酸化来调节常染色质的表观遗传状态，并确定对活性(常染色质)或沉默(异染色质)染色质具有诊断作用的染色质标记的分布变化。为了测试这些表观遗传标记的相互关系，我们将进一步确定靶向组蛋白修饰的组合是否可以抵消或增强单一修饰引起的染色质结构变化。2)在第二个目标中，我们将检验这一假设，即表观遗传的H3S10磷酸化足以抵消异型传播和沉默，而不依赖于多线染色体形态的严重变化。我们将使用PEV抑制/增强作为“读出”H3S10磷酸化对基因表达的相对影响。为了分离出JIL-1的结构和催化作用，我们将在野生型和JIL-1缺失突变背景中转基因表达截短型和“激酶死亡”的JIL-1蛋白，并量化不同报告对PEV的影响。3)在第三个目标中，我们将解决H3S10磷酸化是否针对特定基因组位置的问题。我们将通过不分割唾液腺染色体的芯片序列来特异地定位间期JIL-1和H3S10磷酸化位点来回答这个问题。我们将使用微阵列分析来确定其表达水平受JIL-1介导的H3S10磷酸化调控的基因。我们预计，JIL-1对H3S10磷酸化的拟议研究将极大地扩展我们对特定表观遗传标记如何调控染色质结构和基因调控的了解，这是一个与人类发育和包括癌症在内的疾病直接相关的话题。 公共卫生相关性： 项目简介拟议的实验将极大地提高我们对H3S10磷酸化标记控制异染色质形成和表观遗传基因调控的分子机制的理解。基因沉默是一个关键的发育过程，与包括癌症在内的许多人类健康问题有关。

DESCRIPTION (provided by applicant): Project Summary/Abstract A number of molecules with critical roles in chromatin organization, remodeling, and epigenetic modification have been identified in recent years. However, our understanding of which ones are the key regulators or how chromatin structure marked by a specific epigenetic modification is established is far from complete. We have presented evidence that epigenetic histone H3S10 phosphorylation by the JIL-1 kinase at interphase is a key regulator of euchromatic regions by antagonizing heterochromatization and gene silencing in Drosophila. Consequently, to understand regulation of heterochromatin formation and gene silencing in Drosophila, a premier model system for such studies, it will be crucial to determine the molecular mechanisms of the H3S10ph mark's role in this process. Based on our previous findings we propose a model where JIL-1 kinase activity and phosphorylation of histone H3S10 functions to antagonize heterochromatization by regulating a dynamic balance between factors promoting repression and activation of gene expression. The molecular mechanisms underlying this hypothesis will be explored in three specific aims: 1) In the first aim we will test the hypothesis that H3S10 phosphorylation functions to regulate the epigenetic state of euchromatin by using a LacI-tethering system to ectopically induce H3S10 phosphorylation and determine the changes in the distribution of chromatin markers that are diagnostic for active (euchromatic) or silenced (heterochromatic) chromatin. In order to test the inter-relationship of these epigenetic marks, we will furthermore determine whether combinations of targeted histone modifications can counteract or enhance changes in chromatin structure caused by the single modification. 2) In the second aim we will test the hypothesis that epigenetic H3S10 phosphorylation is sufficient to counteract heterochomatic spreading and silencing independently of gross alterations in polytene chromosome morphology. We will use PEV suppression/enhancement as a "read out" for the relative influence of H3S10 phosphorylation on gene expression. To separate out structural and catalytic contributions of JIL-1 we will express truncated and "kinase-dead" JIL-1 proteins transgenically in both wild-type and JIL-1 null mutant backgrounds and quantify the effect on PEV of different reporters. 3) In the third aim we will address the question of whether H3S10 phosphorylation is targeted to specific genomic locations. We will answer this question by specifically mapping interphase JIL-1 and H3S10 phosphorylation sites by ChIP-seq of non-dividing salivary gland chromosomes. We will use microarray analysis to identify genes whose expression levels are regulated by JIL-1-mediated H3S10 phosphorylation. We expect that the proposed studies of H3S10 phosphorylation by JIL-1 will greatly extend our knowledge of how a specific epigenetic mark modulates chromatin structure and gene regulation, a topic that is directly relevant to human development and disease including cancer. PUBLIC HEALTH RELEVANCE: Project Narrative The proposed experiments will greatly enhance our understanding of the molecular mechanisms controlling heterochromatin formation and epigenetic gene regulation by the H3S10 phosphorylation mark. Gene silencing is a critical developmental process relevant to many human health problems that include cancer.