Understanding the roles of histone demethylation in germline maintenance

描述(由申请人提供)：组蛋白尾巴是各种共价但可逆修饰的目标，这些修饰影响染色质结构，并将特定蛋白质复合体的活动引导到染色体上的特定位置，以实现特殊功能。组蛋白赖氨酸的甲基化在转录和DNA损伤反应中具有功能，在这些缺陷可能导致癌症的地方，也在减数分裂过程中的交叉形成中发挥着特别突出的作用，在减数分裂中，缺陷可能导致各种出生异常、不孕和流产。赖氨酸去甲基酶的去调控或突变与多种癌症有关，突出了它们在维持基因组完整性方面的重要性。线虫中存在三种可能的LSD1/2同源蛋白(SPR-5、AMX-1和T08D10.2)，我们的初步研究表明，这些蛋白对线虫种系的正确维持至关重要，并可能在DNA损伤修复中发挥直接作用。这项建议的目标是建立一种对组蛋白甲基化在减数分裂过程中协调关键染色体事件的方式的机械性理解。我们提出的实验将深入了解去甲基化在促进生殖系减数分裂功能和维持基因组完整性方面所扮演的重要但尚未明确的角色。线虫种系是研究减数分裂和种系维持的理想系统，这是因为整个性腺减数分裂核的独特的时空组织，以及已经开发的一系列工具来跟踪种系中的重要减数分裂事件。此外，鉴于线虫和哺乳动物之间的基因和生物学途径具有高度的同源性，这项工作将对理解高等真核生物的种系维持也非常有用。我们将使用遗传学、生化和细胞学相结合的方法对线虫中的LSD1/2类蛋白进行功能鉴定，并确定其组蛋白去甲基酶活性的特定底物和他们的基因靶标。具体地说，我们建议：1)在体外和体内用生物化学和生物方法确定线虫LSD1/2样蛋白的底物特异性，并确定它们在减数分裂过程中的基因组结合谱；2)通过评估在诱导的DNA损伤时需要什么来重新定位LSD1样蛋白，并确定它们是否是DNA修复机制在损伤部位组装所必需的，从而从机制上深入了解LSD1样蛋白在DNA损伤修复中的作用；以及3)进行有针对性的RNAi筛选，以确定与LSD1/2类蛋白功能相互作用和/或调节其活性的蛋白质，使我们深入了解参与调节组蛋白的蛋白质网络 减数分裂过程中的甲基化状态。这项工作将提供对组蛋白去甲基酶在生殖系维持和减数分裂中的作用的机械性见解，这些作用可能在人类中保留。

DESCRIPTION (provided by applicant): Histone tails are the targets of an enormous variety of covalent but reversible modifications that affect chromatin structure and direct the activities of specific protein complexes to sites along the chromosome for specialized functions. The methylation of histone lysines has a function in transcription and DNA damage response, where defects can result in cancers, and also plays a particularly prominent role in crossover formation during meiosis, where defects can result in a variety of birth abnormalities, infertility and miscarriages. The deregulation or mutation of lysine demethylases has been associated with a variety of cancers, highlighting their importance in maintenance of genomic integrity. Three putative LSD1/2 (human lysine specific demethylase) homologs exist in C. elegans (SPR-5, AMX-1, and T08D10.2), and our preliminary studies indicate that these proteins are important for proper maintenance of the C. elegans germline and may play a direct role for in DNA damage repair. The goal of this proposal is to develop a mechanistic understanding of the way histone methylation orchestrates critical chromosomal events during meiosis. The experiments we propose will provide insight into the important yet poorly-defined roles that demethylation plays in promoting meiotic function and maintenance of genomic integrity in the germline. The C. elegans germline is an ideal system for studying meiosis and germline maintenance due to the unique spatial and temporal organization of meiotic nuclei throughout the gonad and the range of tools that have been developed to track important meiotic events in the germline. Furthermore, given the high degree of homology shared throughout genes and biological pathways between C. elegans and mammals this work will be very informative in understanding germline maintenance in higher eukaryotes as well. We will use combined genetic, biochemical, and cytological approaches to functionally characterize the LSD1/2-like proteins in C. elegans and determine both the specific substrates for their histone demethylase activity and their gene targets. Specifically, we propose: 1) To biochemically and biologically determine the substrate specificity of the C. elegans LSD1/2-like proteins both in vitro and in vivo, and determine their genomic binding profiles throughout meiosis; 2) To gain mechanistic insight into the role of the LSD1-like proteins in DNA damage repair by assessing what is required for them to relocalize in response to induced DNA damage and determining whether they are required for the DNA repair machinery to assemble at sites of damage; and 3) To carry out a targeted RNAi screen to identify proteins that functionally interact with and/or regulate the activity of the LSD1/2-like proteins, giving us insight into the network of proteins involved in regulating histone methylation status during meiosis. This work will provide mechanistic insights into the roles of histone demethylases both in germline maintenance and meiosis that are likely to be conserved in humans.