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

项目摘要/摘要

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.