Temporal regulation of prophase I events by the cyclin-dependent kinases (CDKs) 2 and 4 in mammalian meiosis

项目摘要/摘要 减数分裂是产生单倍体配子的过程，是一个复杂的细胞过程， 染色体必须首先复制，然后经历两轮连续的分裂。第二组是 类似于有丝分裂，姐妹染色单体是分离的。然而，第一个部门是 减数分裂所特有的，涉及同源染色体的分离。精准的分离 染色体是至关重要的--如果不能正确地经历这些过程，可能会导致细胞死亡，或者在更极端的情况下 非整倍体导致唐氏综合症等疾病。减数分裂前期I期发生的事件 为它们的准确分裂准备同源染色体。在第一阶段，DNA双链 断裂形成并修复为互换或非互换，同源染色体找到彼此， 配对、突触和去突触。第一阶段的事件必须严格控制，以便它们发生在 相互之间以及正在进行的其他细胞过程在时间上协调一致的方式。 与有丝分裂细胞周期一样，细胞周期蛋白依赖性激酶(CDKs)复合体调节细胞事件。 前期I，特别是两个CDK，CDK2和CDK4参与了前期I的调节。 CDK2和CDK4定位于减数分裂前期I不同时期的染色体和突变 除了成年鼠缺乏减数分裂细胞外，无论是小鼠的CDK还是小鼠的CDK都基本正常。三、分析了 然而，由于缺乏前期I，CDK2或CDK4驱动的调节的磷酸化靶标被排除在外 比较细胞内蛋白质的磷酸化状态。鉴于这些结果，我假设CDK2和CDK4在 在前期I期间时间定义的时间段，以协调DSB修复和突触的事件 细胞周期的限制。这项建议中的研究旨在阐明这些因素的调节作用和目标 CDKs前期I在目标1中，我将研究小鼠细胞周期蛋白-CDK的特异性相互作用和调控 利用酵母系统进行减数分裂前期I。在目标2中，我将通过以下方式产生新的CDK2和CDK4突变小鼠系 CRISPR/Cas9允许选择性地抑制激酶活性和识别特定的蛋白质靶标。在……里面 目标3我将使用抑制性CDK2和CDK4小鼠系统，以及其他技术来同步 雄性小鼠体内的异步生精细胞群。总体而言，该项目将考虑到时间 识别前期I特异性细胞周期蛋白-CDK驱动的靶点及其调控，这是实现 了解第一阶段的协调活动。

Project Summary / Abstract Meiosis, the process by which haploid gametes are generated, is a complex cellular process whereby chromosomes must first replicate then undergo two successive rounds of division. The second division is similar to that of the mitotic division, where sister chromatids are segregated. The first division however, is unique to meiosis and involves the segregation of homologous chromosomes. The accurate segregation of chromosomes is critical – failure to undergo these correctly can lead to cell death, or in more extreme cases aneuploidy leading to disorders such as Down Syndrome. The events that occur during meiotic prophase I prepare the homologous chromosomes for their accurate division. During prophase I, DNA double-strand breaks are formed and repaired as crossovers or non-crossovers, homologous chromosomes find each other, pair, synapse and desynapse. The events of prophase I must be exquisitely controlled such that they occur in a coordinated temporally manner in relation to each other and the other cellular processes being undertaken. As with the mitotic cell cycle, cyclin-cyclin dependent kinase (CDKs) complexes regulate the cellular events of prophase I. Specifically two CDKs, CDK2 and CDK4, have been implicated in the regulation of prophase I. CDK2 and CDK4 localize to meiotic chromosomes at different temporal stages of prophase I and mutation of either CDK in mouse leads to largely normal mice except for a lack of meiotic cells in the adult. Analysis of the phosphorylated targets of CDK2 or CDK4 driven regulation is however precluded due to a lack of prophase I cells to compare protein phosphorylation status. Given these results I hypothesize that CDK2 and CDK4 act at temporally defined periods during prophase I to co-ordinate events of DSB repair and synapsis, within the confines of the cell cycle. Studies in this proposal aim to elucidate the regulatory roles and targets of these CDKs prophase I. In aim 1 I will investigate the cyclin-CDK specific interactions and regulation in mouse meiotic prophase I using yeast systems. In aim 2, I will generate novel CDK2 and CDK4 mutant mouse lines by CRISPR/Cas9 allowing for selective inhibition of kinase activity and identification of specific protein targets. In aim 3 I will use the inhibitory CDK2 and CDK4 mouse systems, in addition to other techniques, to synchronize the asynchronous germ cell populations in male mice. Overall this project will allow for the temporal identification of prophase I specific cyclin-CDK driven targets and their regulation, a critical step to understanding the coordinated events of prophase I.