Regulation of MutL-gamma function in mediating crossing over in mammalian meiosis

在减数分裂前期，同源重组(HR)是由数百个DNA双链断裂(DSB)启动的，导致交叉(CO)的形成，这是维持染色体相互作用直到第一次减数分裂所必需的。在小鼠中，只有不到10%的DSB成为CoS，但在细胞周期开始之前，这些事件的放置和时间必须精确地安排好。对此至关重要的是顺序加载CO形成所需的两个杂二聚体：MutSg(MSH4/MSH5)早期加载到~150个DSB上，然后是MutLg(MLH1/MLH3)，它被招募到这些MutSg许可的站点中的~23个。PI的实验室在阐明MutSg和MutLg在指定哺乳动物CO命运中的作用方面发挥了核心作用，我们最近确定Cyclin N末端结构域包含-1(CNTD1)对于削减MutSg位点和加载MutLg以产生准确的CO数值是必不可少的。出乎意料的是，我们发现哺乳动物的CNTD1并不作为细胞周期蛋白发挥作用，而是通过招募两个主要的复合体来执行其功能。第一个是复制因子C(RFC)，它将两个夹子加载到DNA上(增殖细胞核抗原和9-1-1)。在体细胞中，增殖细胞核抗原激活MLH1，而9-1-1是小鼠减数分裂过程中必不可少的检查点调节因子。第二个复合体是Skp1-cullin 1-Fbox复合体(SCF)，这是一种泛素E3连接酶，其主要靶点是WEE1，它通过磷酸化使CDK1和CDK2失活。CDK1的磷酸化通过失活成熟促进因子来阻止中期I的进展。我们推测，CNTD1作为一种监测机制，通过其与RFC的相互作用来监测CO的指定和成熟，然后通过其与SCF的相互作用来促进细胞周期的进展，但只有在确保CO形成的情况下才能起作用。本研究的目的有三个：(1)阐明CNTD1调控RFC钳夹和增殖细胞核抗原/9-1-1钳夹的选择和使用以协调I类CO的命名和成熟的机制。我们将阐明RFC、增殖细胞核抗原和其他钳位/钳位加载器组合在促进CNTD1下游的CO和检查点控制中的作用。(2)探讨SCF在第I类一氧化碳调节和细胞周期进程中的CNTD1依赖性作用。这一目标的研究将确定减数分裂SCF的组成，CNTD1如何通过其在细胞质中的相互作用来调控SCF，以及CNTD1/SCF如何调控细胞周期进程。(3)了解哺乳动物减数分裂对CNTD1的空间和功能要求。计算机分析表明，CNTD1的周期蛋白结构域在真核生物中的使用情况各不相同，这表明CNTD1可以通过两种不同的方式发挥作用，以促进I期前期进展(作为细胞周期蛋白，招募CDK，或者作为RFC和SCF的调节因子)。我们将通过在小鼠身上驱动任何一种形式的表达来探索这种双重活动。更多的研究将调查CNTD1在早期阶段细胞间的分布和移动。这些遗传学方法将阐明哺乳动物CNTD1蛋白如何在早期阶段协调CO指定。

During prophase I of meiosis, homologous recombination (HR) is initiated by the formation of hundreds of DNA double strand breaks (DSBs), leading to the formation of crossovers (CO) that are essential for maintaining chromosome interactions until the first meiotic division. In mouse, less than 10% of DSBs become COs, but the placement and timing of these events must be orchestrated with exact fidelity before the cell cycle can commence. Critical to this is the sequential loading of two heterodimers required for CO formation: MutSg (MSH4/MSH5) loads early onto ~150 DSBs, followed by MutLg (MLH1/MLH3) which is recruited to ~23 of these MutSg-licensed sites. The PI’s lab has been a central player in elucidating the roles of MutSg and MutLg in designating a CO fate in mammals, and we recently determined that Cyclin N-terminal Domain- containing-1 (CNTD1) is essential for paring down MutSg sites and loading MutLg to generate precise CO numbers. Unexpectedly, we find that mammalian CNTD1 does not act as a cyclin, and instead executes its functions by recruiting two major complexes. The first is Replication Factor C (RFC) which loads two clamps onto DNA (PCNA and 9-1-1). In somatic cells, PCNA activates MLH1, while 9-1-1 is an essential checkpoint regulator in mouse meiosis. The second complex is the SKP1-Cullin 1-Fbox complex (SCF), a ubiquitin E3 ligase whose major target is WEE1, which inactivates CDK1 and CDK2 through phosphorylation. Phosphorylation of CDK1 prevents metaphase I progression by inactivating Maturation Promoting Factor. We hypothesize that the CNTD1 acts as a surveillance mechanism to monitor CO designation and maturation through its interactions with RFC, and then to promote cell cycle progression through its interactions with SCF, but only once CO formation is assured. Three aims are proposed: (1) to elucidate the mechanism by which CNTD1 regulates the choice and usage of RFC clamp loaders and the PCNA/9-1-1 clamps to orchestrate class I CO designation and maturation. We will elucidate the role of RFC, PCNA, and other clamp/clamp loader combinations in promoting CO and checkpoint control downstream of CNTD1. (2) To explore the CNTD1-dependent role of the SCF in class I CO regulation and cell cycle progression. Studies in this aim will determine the components of the meiotic SCF, how CNTD1 regulates SCF through its interactions in the cytoplasm, and how CNTD1/SCF regulates cell cycle progression. (3) To understand the spatial and functional requirements for CNTD1 in mammalian meiosis. In silico analysis suggests variable use of the cyclin domain of CNTD1 across eukaryotes, suggesting that CNTD1 can function in two distinct ways to facilitate prophase I progression (as a cyclin, recruiting CDKs, or as a regulator of RFC and SCF). We will explore this dual activity by driving expression of either form in the mouse. Additional studies will investigate the distribution and movement of CNTD1 between cellular compartments during prophase I. These genetic approaches will elucidate how the mammalian CNTD1 protein functions to orchestrate CO designation during prophase.