Meiotic checkpoint pathways and gametocyte quality control

 描述（由申请方提供）：雌性的生殖寿命取决于静息卵母细胞池的大小。卵母细胞的过度闭锁发生在减数分裂前期的缺陷和外源性DNA损伤，如癌症放疗和化疗。例如，小鼠Spo 11突变体中的卵母细胞池在三个月内被消除，因为Spo 11-/-不能启动重组和配对同源染色体。在Msh 4突变体中，重组开始，但重组和同源配对/突触的进展是有缺陷的。Msh 4-/-的卵母细胞池在出生后4天内耗尽。推断减数分裂检查点途径的两个不同分支分别响应于重组或同源配对中的缺陷。DNA损伤反应（DDR）的几个组成部分定义了减数分裂对未修复重组中间体的反应。相反，对有缺陷的突触的反应需要减数分裂特异性因子HORMAD 1，并通过称为未配对染色质减数分裂沉默（MSUC）的过程引起转录沉默。 我已经确定RNF 212是减数分裂检查点机制的一个新组分。引人注目的是，Rnf 212突变恢复了Spo 11-/-和Msh 4-/-背景下的休眠卵母细胞池。有趣的是，Rnf 212编码RING家族E3连接酶，该酶催化小泛素样分子SUMO对蛋白质的修饰。这些数据表明，RNF 212介导的SUMO化是导致卵母细胞凋亡的减数分裂检查点信号通路的一个关键方面。 我将结合遗传学、细胞生物学和分子生物学的方法研究RNF 212依赖的小鼠卵母细胞质量控制的性质和机制。这些研究将为我自己的实验室的研究奠定基础，这些研究将追求对配子质量控制的机械理解。 目的1是确定卵母细胞的救援效率突触/重组突变体和RNF 212是否有助于生理产后闭锁。在Spo 11-/-、Spo 11-/-Rnf 212-/-、Msh 4-/-和Msh 4-/-Rnf 212-/-突变体背景中，将对新生儿和不同年龄动物卵巢中的卵母细胞和发育卵泡总数进行定量。 目的2是了解Rnf 212突变如何拯救突触/重组突变体中的卵母细胞池。许多模型可以解释Rnf 212突变如何绕过突触和重组突变体中的卵母细胞损失。为了开始区分这些模型，我将进行胎儿卵巢减数分裂前期的详细表征。 目的3是确定RNF 212介导的SUMO化参与减数分裂检查点的靶点。了解RNF 212在减数分裂检查点信号传导中的作用将需要鉴定相关的靶蛋白。在这个目标中，我将通过候选和无偏的方法，使用蛋白质印迹，酵母双杂交和蛋白质组学方法的组合来确定RNF 212的目标。

 DESCRIPTION (provided by applicant): Reproductive lifespan in females relies on the size of the resting oocyte pool. Excessive atresia of oocytes occurs in response to defects in meiotic prophase and extraneous DNA damage such as cancer radiotherapy and chemotherapy. For instance, oocyte pools in mouse Spo11 mutants are eliminated within three months because Spo11-/- fails to initiate recombination and pair up homologous chromosomes. In Msh4 mutants, recombination is initiated, but progression of recombination and homolog pairing/synapsis are defective. The oocyte pools of Msh4-/- are depleted within four days of birth. Two distinct branches of the meiotic checkpoint pathway are inferred to respond to defects in recombination or homolog pairing, respectively. Several components of the DNA damage response (DDR) define the meiotic response to unrepaired recombination intermediates. In contrast, the response to defective synapsis requires a meiosis-specific factor, HORMAD1, and provokes transcriptional silencing via a process termed Meiotic Silencing of Unpaired Chromatin (MSUC). I have identified RNF212 as a novel component of the meiotic checkpoint machinery. Strikingly, Rnf212 mutation restores the resting oocyte pools in both Spo11-/- and Msh4-/- backgrounds. Intriguingly, Rnf212 encodes an RING-family E3-ligase that catalyzes a protein modification by the Small Ubiquitin-like Molecule, SUMO. These data imply that RNF212-mediated SUMOylation is a key aspect of the meiotic checkpoint-signaling pathway that leads to oocyte apoptosis. I will investigate the nature and mechanism of RNF212-dependent oocyte quality control in mouse using a combination of genetics, cell biology and molecular approaches. These studies will build the foundation for research in my own laboratory that will pursue a mechanistic understanding of gamete quality control. Aim1 is to determine the efficiency of oocyte rescue in synapsis/recombination mutants and whether RNF212 contributes to physiological post-partum atresia. In the Spo11-/-, Spo11-/-Rnf212-/-, Msh4-/-, and Msh4-/-Rnf212-/- mutant backgrounds, total numbers of oocytes and developing follicles will be quantified in the ovaries of newborns and animals of various ages. Aim 2 is to understand how Rnf212 mutation rescues oocyte pools in synapsis/recombination mutants. A number of models could explain how Rnf212 mutation can bypass oocyte loss in synapsis and recombination mutants. To begin to distinguish between these models, I will perform a detailed characterization of meiotic prophase in fetal ovaries. Aim 3 is to identify targets of RNF212-mediated SUMOylation involved in the meiotic checkpoint. Understanding the role of RNF212 in meiotic checkpoint signaling will require identification of pertinent target proteins. In this aim, I will identify RNF212 targets through a combination of candidate and unbiased approaches using western blot, yeast-two-hybrid and proteomics approaches.