睾丸sORF编码蛋白TSEP7的功能研究

近年来，研究发现随着年龄的增长，DNA损伤可以在雄性生殖细胞中逐渐累积，造成精子发生障碍、雄性配子染色体损伤、子代异常等一系列症状。因此对于雄性生殖细胞染色质动态调节以及损伤保护的分子机制探索，对于阐明部分男性不育的发病机制十分重要。在申请人课题组的前期研究中建立了核糖体印迹与蛋白质组学相结合的睾丸sORF编码蛋白鉴定流程，发现TSEP7在细胞核内可以出现斑点状定位。通过CRISPR/Cas9技术构建了TSEP7敲除小鼠，应用白消安制造基因毒性损伤模型，发现TSEP7敲除小鼠较野生型小鼠的精子发生恢复存在显著障碍，导致不育。本项目拟通过TSEP7敲除小鼠损伤模型和衰老模型深入分析，明确TSEP7缺失对于雄性生殖细胞的影响；进一步通过ChIP-seq揭结合ATAC-seq技术等揭示TSEP7缺失对染色质的影响，进而阐明小蛋白TSEP7调控雄性生殖细胞染色质结构的机制。

In recent years, it has been found that with the increase of age, DNA damage can gradually accumulate in male germ cells, resulting in a series of symptoms such as spermatogenesis disorder, damage of male gamete chromosomes, abnormal offspring. Therefore, the exploration of the molecular mechanism of chromatin dynamic regulation and damage protection of male germ cells can help to clarify the pathogenesis of some male infertility. In the preliminary study of the applicant's research group, a process of identifying the testicular sORF encoded proteins(TSEPs) was established, which combined ribosome footprint with proteomics. An evolutionarily conserved TSEP among human and mouse, TSEP7, was found. The localization of TSEP7 in the nucleus was spot-like. TSEP7-DNA mixture could form spherical droplets in vitro. Using CRISPR/Cas9, TSEP7 knockout mice were constructed. TSEP7 knockout mice had significant defects in recovery from busulfan genotoxic stress, resulting in infertility. In this project, we will analyze busulfan genotoxic stress and aging of TSEP7 knockout mice, to test the function of TSEP7 in vivo. The changes of chromatin will be revealed by ChIP-seq and ATAC-seq. Through the above experiments, the role of TSEP7 in the chromatin regulation of male germ cells will be revealed.