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

项目概要/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，然后进行连续两轮的分裂。第二个部门是 类似于有丝分裂，姐妹染色单体分离。然而，第一个部门是 减数分裂所特有的，并涉及同源染色体的分离。精确分离 染色体是至关重要的，如果不能正确地进行这些操作，可能会导致细胞死亡，或者在更极端的情况下， 非整倍体导致疾病，如唐氏综合症。减数分裂前期I发生的事件 为同源染色体的精确分裂做好准备。在前期I，DNA双链 断裂在交换或非交换时形成并修复，同源染色体找到彼此， 对、突触和去突触。前期I的事件必须被精细地控制，使得它们发生在 一种时间上相互协调的方式，以及其他正在进行的细胞过程。 与有丝分裂细胞周期一样，细胞周期蛋白-细胞周期蛋白依赖性激酶（CDKs）复合物调节细胞周期的细胞事件。 前期Ⅰ特别是两种CDK，CDK 2和CDK 4，已经涉及前期I的调节。 CDK 2和CDK 4定位于减数分裂前期Ⅰ的不同时间阶段的染色体上， 小鼠中的任一种CDK导致除了成年小鼠中缺乏减数分裂细胞之外的基本上正常的小鼠。分析 然而，由于缺乏前期I，CDK 2或CDK 4驱动的调节的磷酸化靶被排除。 细胞比较蛋白磷酸化状态。鉴于这些结果，我假设CDK 2和CDK 4在 在前期I期间的时间上定义的时期，以协调DSB修复和突触的事件，在 细胞周期的界限。本提案中的研究旨在阐明这些基因的调节作用和靶点， CDK前期I。目的1研究细胞周期蛋白与CDK的特异性相互作用及其调控机制 使用酵母系统的减数分裂前期I。在目标2中，我将通过以下方法产生新的CDK 2和CDK 4突变小鼠系： CRISPR/Cas9允许选择性抑制激酶活性和鉴定特异性蛋白质靶标。在 目的3我将使用抑制性CDK 2和CDK 4小鼠系统，以及其他技术， 雄性小鼠的异步生殖细胞群。总的来说，这个项目将允许时间 鉴定前期I特异性细胞周期蛋白-CDK驱动靶点及其调节， 了解前期I的协调事件。

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.