Understanding the roles of histone demethylation in germline maintenance

描述（由申请人提供）：组蛋白尾部是大量共价但可逆修饰的靶标，这些修饰影响染色质结构并将特定蛋白质复合物的活性引导至染色体沿着位点以实现特定功能。组蛋白赖氨酸的甲基化在转录和DNA损伤反应中发挥作用，其中缺陷可能导致癌症，并且在减数分裂期间的交叉形成中也发挥着特别突出的作用，其中缺陷可能导致各种出生异常、不孕和流产。赖氨酸脱甲基酶的失调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。在C. elegans（SPR-5，AMX-1，和T08D10.2），并且我们的初步研究表明这些蛋白对于C.可能在DNA损伤修复中起直接作用。这个提议的目标是发展一个机制的理解组蛋白甲基化编排关键染色体事件在减数分裂过程中的方式。我们提出的实验将深入了解去甲基化在促进生殖细胞减数分裂功能和维持基因组完整性方面所起的重要但定义不清的作用。梭由于整个生殖腺中减数分裂核的独特空间和时间组织以及已经开发的用于追踪生殖系中重要减数分裂事件的工具范围，线虫生殖系是研究减数分裂和生殖系维持的理想系统。此外，由于C.这一工作对于理解高等真核生物的生殖系维持也将是非常有益的。 我们将结合遗传学、生物化学和细胞学的方法对C. elegans和确定其组蛋白去甲基化酶活性的特异性底物和其基因靶标。具体而言，我们提出：1）生物化学和生物学确定的底物特异性C。2）通过评估LSD 1样蛋白在诱导的DNA损伤中重新定位所需的条件，并确定它们是否是DNA修复机制在损伤位点组装所需的条件，从而获得对LSD 1样蛋白在DNA损伤修复中的作用的机理认识;和3）进行靶向RNAi筛选以鉴定与LSD 1/2样蛋白功能性相互作用和/或调节LSD 1/2样蛋白活性的蛋白质，使我们深入了解参与调节组蛋白的蛋白质网络 甲基化状态。这项工作将提供机制的见解组蛋白去甲基化酶的作用，无论是在生殖细胞的维护和减数分裂，可能是保守的人类。

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.