Meiotic checkpoint pathways and gametocyte quality control

 描述(申请人提供)：雌性的生殖寿命取决于休眠卵母细胞池的大小。卵母细胞过度闭锁是对减数分裂前期缺陷和外来DNA损伤的反应，如癌症、放射治疗和化疗。例如，小鼠Spo11突变体中的卵母细胞池在三个月内就被消除了，因为Spo11-/-未能启动重组并将同源染色体配对。在Msh4突变体中，重组被启动，但重组和同源配对/突触的进展是有缺陷的。Msh4-/-的卵母细胞池在出生后四天内就会耗尽。减数分裂检查点途径的两个不同分支被推测分别对重组或同源配对中的缺陷做出反应。DNA损伤反应(DDR)的几个组成部分定义了减数分裂对未修复的重组中间体的反应。相反，对有缺陷的突触的反应需要一个减数分裂特异性因子HORMAD1，并通过一种称为未配对染色质的减数分裂沉默(MSUC)的过程来引发转录沉默。我已经确定RNF212是减数分裂检查点机制的一个新组件。引人注目的是，Rnf212突变恢复了Spo11-/-和Msh4-/-背景中的静止卵母细胞池。有趣的是，Rnf212编码了一个环状家族的E3-连接酶，它催化了类似泛素的小分子SUMO对蛋白质的修饰。这些数据表明，RNF212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，对RNF212依赖的小鼠卵母细胞质量控制的性质和机制进行研究。这些研究将为我自己的实验室的研究奠定基础，我将追求对配子质量控制的机械理解。目的：探讨突触/重组突变体拯救卵母细胞的效率以及RNF212是否与生理性产后闭锁有关。在Spo11-/-、Spo11-/-Rnf212-/-、Msh4-/-和Msh4-/-Rnf212-/-突变背景中，不同年龄的新生儿和动物的卵巢中卵母细胞和发育中的卵泡的总数将被量化。目的2是了解Rnf212突变是如何挽救突触/重组突变体中的卵母细胞池的。许多模型可以解释Rnf212突变如何在突触和重组突变体中绕过卵母细胞的损失。为了开始区分这些模型，我将对胎儿卵巢的减数分裂前期进行详细的描述。目的3是确定参与减数分裂检查点的RNF212介导的SUMO化的靶点。要了解RNF212在减数分裂检查点信号中的作用，需要识别相关的靶蛋白。在这个目标中，我将通过使用蛋白质印迹、酵母双杂交和蛋白质组学方法，通过候选和公正的方法相结合的方法来识别RNF212靶点。

 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.