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

描述（由申请人提供）：减数分裂前期I期间的交换对于将同源染色体拴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下的交叉（CO）的定位对于确保同源染色体在MI处的平等分离是至关重要的，其重要性通过以下观察而得到强调：人类中所有自发流产的50%是由于该阶段的染色体错误分离错误。因此，毫不奇怪，CO的形成受到严格的调节，使得在早期前期I期间出现的10倍过量的起始双链断裂（DSB）事件中，实现了非常少的最终CO。DSB中间体的CO命名主要由“ZMM”基因控制，所述“ZMM”基因包括减数分裂特异性DNA错配修复异二聚体复合物MutSc（MSH 4/MSH 5）和MutLc（MLH 1/MLH 3）。MutSc首先与250+ DSB中的150个结合，其中约24-28个随后积累MutLc成为I类CO。MutSc位点的这个特定子集是如何指定的尚不清楚，但我们实验室最近的研究表明，含有细胞周期蛋白N末端结构域的蛋白质-1（CNTD 1）在这一过程中发挥着至关重要的作用，因为MutSc焦点频率在整个前期保持持续升高在带有Cntd 1基因突变的小鼠突变体中。此外，MutLc未能加载在Cntd 1突变体的后期前期I染色体，这表明CO指定和成熟密切耦合的CNTD 1调控的事件。连同其他调节交叉指定，包括Zip 3/ZHP- 3的直系同源物，RNF 212，和人类入侵增强子-10（HEI 10），我们假设CNTD 1的行为，以确保交叉指定在I类CO途径，通过促进加工/成熟的有限集MutSc事件，或通过去除多余的DSB修复中间体。本研究的目的是阐明这种新的交叉指定调节电路。在目标1中，我们将询问如何以及何时使用新的Cntd 1-V5标记的小鼠将CNTD 1招募到减数分裂染色体核心，并通过评估在MutSc水平降低的情况下CNTD 1的定位。在目标2中，我们将确定介导CNTD 1功能的关键功能相互作用，并且我们将确定CNTD 1是否与一种或几种细胞周期蛋白依赖性激酶协同作用，因为其作为细胞周期蛋白家族成员的地位将表明。在目标3中，我们将阐明CNTD 1丢失导致尽管存在持续的MutSg复合物但不存在MutLg病灶的机制，询问小鼠基因组中CO的指定是否需要特定DSB中间位点处的剂量依赖性MutSc阈值水平，其积累和/或稳定性通过CNTD 1的加载和活性来确保。这些研究利用了PI实验室中令人印象深刻的小鼠突变体库，以及利用小鼠转基因和哺乳动物生殖细胞中前期I事件的高分辨率三维成像的创新实验方法。

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.