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期间分离。同源基因的分离需要它们之间的物理联系，这种联系通过交叉发生。交叉是通过一种特殊的重组过程产生的，该过程始于在第一次减数分裂前期程序性地产生DNA双链断裂(DSB)。沿着每条染色体的蛋白质核心/轴产生多个DSB。DSB驱动同系物的配对，从而导致同系物之间的突触。DSB的修复也产生了同源基因间的交叉。鉴于交叉的重要性和DSB的潜在遗传毒性，对DSB的形成进行了严格的控制。因此，DSB只能在未联结的轴上形成，在那里需要DSB来促进同源突触。我们最近的工作确定IHO1是一种与非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向轴的招募在很大程度上取决于IHO1的S与未突触的轴传感器HORMAD1的相互作用。因此，我们假设IHO1-HORMAD1相互作用是将DSB形成限制在非突触轴的机制的关键。我们的数据表明，IHO1-HORMAD1相互作用的调节和由此导致的IHO1轴定位的调节有助于抑制(1)在突触染色体上的DSB形成，(2)在现有的DSB断裂附近，(3)在终止同源性搜索的晚期前期。因此，IHO1似乎是保护胚系免受过度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，并讨论针对IHO1的调控机制的分子基础。主要目的之一是解决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来产生突变的IHO1版本的小鼠，这些突变版本的小鼠在IHO1-HORMAD1相互作用和IHO1轴关联方面存在选择性缺陷。我建议对这些小鼠进行表型分析，以及影响IHO1和HORMAD1功能和调节的其他减数分裂突变的表型分析。我们将把这与IHO1的相互作用研究结合起来，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。鉴于正确控制重组是维持生殖系基因组完整性所必需的，而且重组受损会导致人类非整倍体，预期结果将产生很高的影响，与人类生殖健康明显相关。

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.