Understanding the Role of Histone Demethylases and Heterochromatin in Cell Cycle

描述（由申请人提供）： 抽象的。基因组的不稳定性是癌症等与年龄有关的疾病的发展和发病的一个主要因素。DNA复制的时空时序的异常调节是涉及基因组不稳定性和癌症风险增加的关键因素。染色质结构在协调细胞周期G1期和S期DNA复制的时间方面起着重要作用。染色质是一种高度有序的结构，包含DNA，组蛋白和其他染色体蛋白质。组蛋白的翻译后修饰影响许多生物学过程的结果，包括转录、复制和染色体维持。组蛋白3赖氨酸9三甲基化（H3 K9 me 3）被认为是异染色质的标志，异染色质是一种在基因组中重复和端粒区域发现的抑制结构。H3 K9 me 3和异染色质水平的改变导致异常的复制时间和基因组不稳定性和癌症的发生。了解甲基化和异染色质形成和维持的动态在整个细胞周期中是如何调节的，对于揭示控制复制时机的基本原理至关重要，但最重要的是，确定可能导致癌症中观察到的复制时机缺陷和基因组不稳定性的调节因素。我们最近鉴定了JMJD 2A H3 K9/36三去甲基酶作为染色质可及性、异染色质（HP 13）、DNA复制定时和S期进展的调节剂。我们还证明了JMJD 2A水平和定位通过泛素化在细胞周期中调节，这直接影响JMJD 2A在S期进展中的作用。这些数据令人兴奋，因为调节泛素化已成为癌症治疗的一种治疗策略。我们假设HP 13和JMJD 2A的泛素化调节特定基因组区域的靶向，从而发生有序的细胞周期进程。这项拨款提案将确定泛素化对JMJD 2A细胞周期功能的影响（目标1），并确定JMJD 2A如何改变染色质结构，以便DNA复制时间在空间和时间上得到适当的调节（目标2）。在目标1中，我们将使用蛋白质组学和生物化学方法来确定JMD 2A内修饰的位点/区域、参与添加和去除泛素的酶以及它们对JMD 2A依赖性S期进展的影响。在目标2中，我们将使用基因组学来确定在整个细胞周期中由JMJD 2A和异染色质调节的区域，以及确定JMJD 2A对复制时机的影响（即，引发和/或延伸）。这些研究将显著影响我们对S期进展的基本理解和对JMJD 2A过表达肿瘤的临床理解。 公共卫生相关性： 公共卫生相关性。肿瘤的发生是由异常的染色质结构和DNA复制的时空时序不当引起的。这项资助的重点是了解染色质结构的改变如何影响DNA复制的时间。我们将使用组蛋白去甲基化酶JMJD 2A来解决这种关系，因为JMJD 2A的表达增加会导致染色质可及性增加和DNA复制加快。我们的研究结果将为JMJD 2A水平增加的肿瘤提供分子见解，并揭示新的治疗策略。

DESCRIPTION (provided by applicant): Abstract. Genomic instability is a major contributing factor to the development and onset of age-related diseases such as cancer. Aberrant regulation of the spatial-temporal timing of DNA replication is a critical factor involved in genomic instability and increased cancer risk. Chromatin structure plays a fundamental role in coordinating the timing of DNA replication during G1 and S phases of cell cycle. Chromatin is a highly ordered structure that contains DNA, histones, and other chromosomal proteins. Posttranslational modifications of histones influence the outcomes of numerous biological processes including transcription, replication, and chromosome maintenance. Histone 3 lysine 9 tri-methylation (H3K9me3) is considered a hallmark of heterochromatin, a repressive structure found at repetitive and telomeric regions in the genome. Alterations in H3K9me3 and heterochromatin levels result in aberrant replication timing and the onset of genomic instability and cancer. Understanding how the dynamics of methylation and heterochromatin formation and maintenance are regulated throughout cell cycle is critical for uncovering both basic principals governing replication timing, but most importantly, identifying regulatory factors that could contribute to replication timing defects and genomic instability observed in cancer. We have recently identified the JMJD2A H3K9/36 tri-demethylase as a regulator of chromatin accessibility, heterochromatin (HP13), DNA replication timing and S phase progression. We also demonstrated that JMJD2A levels and localization are regulated over cell cycle by ubiquitination, which directly impacts the role of JMJD2A in S phase progression. These data are exciting since regulating ubiquitination has emerged as a therapeutic strategy in cancer treatment. We hypothesize that HP13 and the ubiquitination of JMJD2A regulate the targeting of specific genomic regions so that orderly cell cycle progression occurs. This grant proposal will determine the impact ubiquitination has on JMJD2A cell cycle function (Aim 1) and determine how JMJD2A alters chromatin structure so that DNA replication timing is properly spatially and temporally regulated (Aim 2). In aim 1, we will use proteomic and biochemical approaches to determine the sites/regions that are modified within JMJD2A, the enzymes involved in adding and removing the ubiquitin, and their impact on JMD2A- dependent S phase progression. In aim 2, we will use genomics to determine the regions regulated by both JMJD2A and heterochromatin throughout cell cycle as well as determine the impact JMJD2A has on replication timing (i.e., initiation and/or elongation). These studies will significantly impact our basic understanding of S phase progression and clinical understanding of JMJD2A overexpressing tumors. PUBLIC HEALTH RELEVANCE: Public Health Relevance. Tumorigenesis arises from both aberrant chromatin structure and improper spatial-temporal timing of DNA replication. This grant focuses on understanding how alterations in chromatin structure impact DNA replication timing. We will use the histone demethylase JMJD2A to address this relationship because increased expression of JMJD2A results in increased chromatin accessibility and faster DNA replication. Our results will provide molecular insights into tumors with increased JMJD2A levels and reveal new therapeutic strategies.