Regulation of MutL-gamma Function in Mediating Crossing Over in Mammalian Meiosis

描述（由申请人提供）：在减数分裂前期I的杂交对于将同源染色体系在一起直到第一次减数分裂（MI）是必不可少的。在正确的时间和空间频率上定位交叉（CO）对于确保MI同源染色体的平等分离至关重要，观察到人类50%的自然流产是由于染色体在这一阶段的错误分离造成的，这一点很重要。因此，毫不奇怪，CO的形成受到严格调控，因此，在前期I出现的启动双链断裂（DSB）事件的10倍过量中，很少实现最终CO。DSB中间体的CO指定主要由“ZMM”基因控制，该基因包括减数分裂特异性DNA错配修复异二聚体复合物MutSc （MSH4/MSH5）和MutLc （MLH1/MLH3）。MutSc首先结合250+ dsb中的150个，其中约24-28个随后积累MutSc成为I类COs。MutSc位点的这一特定子集是如何被指定的尚不清楚，但我们实验室最近的研究表明，细胞周期蛋白n端结构域蛋白-1 （CNTD1）在这一过程中起着至关重要的作用，因为在携带CNTD1基因突变的小鼠突变体中，MutSc聚焦频率在整个I前期持续升高。此外，在Cntd1突变体的I前期晚期，MutLc无法装载到染色体上，这表明CO的指定和成熟是由Cntd1调节的密切耦合事件。与其他交叉指定的调节因子一起，包括Zip3/ZHP- 3同源物、RNF212和人类入侵-10增强子（HEI10），我们假设CNTD1通过促进有限的MutSc事件的处理/成熟，或通过去除多余的DSB修复中间体，来确保I类CO通路中的交叉指定。本提案的研究旨在阐明这种新颖的交叉指定调节电路。在目的1中，我们将使用一种新型CNTD1 - v5标记的小鼠，并通过评估在MutSc水平降低的情况下CNTD1的定位，询问CNTD1如何以及何时被招募到减数分裂染色体核心。在目标2中，我们将确定介导CNTD1功能的关键功能相互作用，我们将确定CNTD1是否与一个或几个周期蛋白依赖性激酶协同作用，正如其作为周期蛋白家族成员的地位所表明的那样。在目标3中，我们将阐明尽管存在MutSg复合物，但CNTD1的缺失导致MutLg灶缺失的机制，询问小鼠基因组中COs的指定是否需要特定DSB中间位点的MutSc的剂量依赖性阈值水平，其积累和/或稳定性是通过CNTD1的负载和活性来确保的。这些研究利用了PI实验室中令人印象深刻的小鼠突变库，以及利用小鼠转基因技术和哺乳动物生殖细胞I前期事件的高分辨率三维成像技术的创新实验方法。

DESCRIPTION (provided by applicant): Crossing over during meiotic prophase I is essential for tethering homologous chromosomes together until the first meiotic division (MI). The localization of crossovers (CO) at the correct temporal and spatial frequency is critical for ensuring equal segregation of homologous chromosomes at MI, the importance of which is underscored by the observation that 50% of all spontaneous miscarriages in humans are due to chromosome mis-segregation errors at this stage. Not surprisingly, therefore, CO formation is tightly regulated, such that, of the 10-fold excess of initiating double strand break (DSB) events that arise during early prophase I, very few final COs are achieved. CO designation of DSB intermediates is largely controlled by the "ZMM" genes that include the meiosis-specific DNA mismatch repair heterodimeric complexes, MutSc (MSH4/MSH5) and MutLc (MLH1/MLH3). MutSc first binds to 150 of the 250+ DSBs and, of these, approximately 24-28 subsequently accumulate MutLc to become class I COs. How this specific subset of MutSc sites are designated is unclear, but recent studies in our lab have revealed that Cyclin N-terminal domain-containing protein-1 (CNTD1) plays a crucial role in this process, since MutSc focus frequency remains persistently elevated throughout prophase I in mouse mutants bearing a mutation in the Cntd1 gene. Moreover, MutLc fails to load on chromosomes during late prophase I in Cntd1 mutants, suggesting that CO designation and maturation are intimately coupled events regulated by CNTD1. Together with other regulators of crossover designation, including the Zip3/ZHP- 3 ortholog, RNF212, and human enhancer of invasion-10 (HEI10), we hypothesize that CNTD1 acts to ensure crossover designation in the class I CO pathway, either by promoting the processing/maturation of a finite set of MutSc events, or by removing excess DSB repair intermediates. Studies in this proposal are aimed at elucidating this novel crossover designation regulatory circuit. In aim 1, we will ask how and when CNTD1 is recruited to meiotic chromosome cores using a novel Cntd1-V5-tagged mouse, and by assessing CNTD1 localization in the presence of reduced levels of MutSc. In aim 2, we will identify key functional interactions that mediate CNTD1 function and we will determine whether CNTD1 acts in concert with one or several cyclin- dependent kinases, as its status as a cyclin family member would suggest. In aim 3, we will elucidate the mechanism by which loss of CNTD1 results in the absence of MutLg foci despite persistent MutSg complexes, asking whether the designation of COs in the mouse genome requires a dose-dependent threshold level of MutSc at specific DSB intermediate sites, whose accumulation and/or stability is ensured through the loading and activity of CNTD1. These studies take advantage of the impressive repertoire of mouse mutants available in the PI's lab, together with innovative experimental approaches that take advantage of advances in mouse transgenesis and high-resolution 3-dimensional imaging of prophase I events in mammalian germ cells.