The control of meiotic DNA break formation by key chromosome axis components, IHO1 and HORMAD1, in mammals.

单倍体配子的产生需要二倍体生殖细胞的同源染色体在减数分裂的第一次分裂中分离。同源物的分离需要它们之间的物理联系，这是通过交叉发生的。交叉是由一个特殊的重组过程产生的，该过程始于第一个减数分裂前期DNA双链断裂（DSBs）的程序化生成。沿着每条染色体的蛋白核心/轴产生多个dsb。dsb驱动同源物配对，从而导致同源物之间的突触。dsb的修复也会产生同源交叉。鉴于交叉的重要性和DSB潜在的遗传毒性，严格控制DSB的形成。因此，仅允许在非突触轴上形成dsb，而在非突触轴上需要dsb来促进同源突触。我们最近的工作发现IHO1是一种与未突触轴相关的蛋白质，对DSB的形成至关重要。根据我们的模型，IHO1在轴上的招募很大程度上取决于IHO1与非突触轴传感器HORMAD1的相互作用。因此，我们假设IHO1-HORMAD1相互作用是限制DSB形成非突触轴的机制的关键。我们的数据表明，IHO1- hormad1相互作用的调控以及由此产生的IHO1轴定位的调节有助于抑制DSB的形成(1)在突触染色体上，(2)在现有DSB断裂附近，(3)在同源性搜索终止的前期晚期。因此，IHO1似乎是保护种系免受过多DNA断裂形成的机制的核心。我们将测试我们的DSB形成模型和IHO1在其中的功能，我们将解决针对IHO1的调控机制的分子基础。其中一个关键目标是解决IHO1- hormad1相互作用和IHO1轴关联在DSB形成中的重要性。因此，我们使用CRISPR/Cas9产生IHO1突变版本的小鼠，这些突变版本在IHO1- hormad1相互作用和IHO1轴关联中选择性缺陷。我建议对这些小鼠和影响IHO1和HORMAD1功能和调控的其他减数分裂突变体进行表型分析。我们将结合IHO1的相互作用研究，以更深入地了解IHO1的功能和调控。所提出的实验将为DSB形成的时空控制的逻辑和分子基础提供重要的新见解。鉴于需要正确控制重组以维持种系基因组的完整性，并且重组受损会导致人类非整倍体，预期的结果将对人类生殖健康产生重大影响，具有明显的相关性。

Generation of haploid gametes requires that homologous chromosomes (homologues) of diploid germ cells segregate during the first division of meiosis. Segregation of homologues requires physical links between them, which occur via crossovers. Crossovers are generated by a specialized recombination process that starts with programmed generation of DNA double strand breaks (DSBs) in the first meiotic prophase. Multiple DSBs are generated along the proteinaceous core/axis of each chromosome. DSBs drive the pairing of homologues, which results in synapsis between homologues. The repair of DSBs also generates the inter-homologue crossovers. Given the importance of crossovers and the potential genotoxicity of DSBs, tight control is exerted on DSB formation. Hence, DSBs are allowed to form only on unsynpased axes, where DSBs are needed to promote homologue synapsis. Our recent work identified IHO1 as a protein that associates with unsynapsed axis and is crucial for DSB formation. According to our model, IHO1 recruitment to axes largely depends on IHO1´s interaction with an unsynapsed-axis-sensor, HORMAD1. Thus, we hypothesize that IHO1-HORMAD1 interaction is key to the mechanism that limits DSB formation to unsynapsed axes. Our data suggest that regulation of IHO1-HORMAD1 interaction and the resulting modulation of IHO1 axis-localization contribute to the inhibition of DSB formation (1) on synapsed chromosomes, (2) in the vicinity of existing DSB breaks and (3) in advanced prophase stages where homology search is terminated. Thus, IHO1 appears central to mechanisms that protect the germ line from excessive DNA break formation.We will test our model of DSB formation and IHO1 function within it, and we will address the molecular basis of the regulatory mechanisms that target IHO1. One of the key aims is to address the importance of IHO1-HORMAD1 interaction and IHO1 axis association in DSB formation. Hence, we used CRISPR/Cas9 to generate mice with mutant IHO1 versions that are selectively defective in IHO1-HORMAD1 interaction and IHO1 axis association. I propose phenotypic analysis of these mice and that of additional meiotic mutants affecting IHO1 and HORMAD1 functions and regulation. We will combine this with interaction studies of IHO1 to gain deeper understanding of IHO1 functions and regulation.The proposed experiments will provide major new insights into the logic and the molecular basis of the spatiotemporal control of DSB formation. Given that correct control of recombination is needed to maintain genome integrity in the germ line, and that impaired recombination causes human aneuploidies, the expected results will have high impact with a clear relevance to human reproductive health.