组蛋白分子伴侣Hira对小鼠卵母细胞发育的调控机理

Mammalian oocytes go through a long and complex developmental process while acquiring the competencies that are required for fertilization and embryogenesis. Conditional depletion of histone chaperone Hira during oocyte development results in reduction of ovarian size and extensive oocyte death. However, the underlying molecular mechanism remains elusive. In the proposed project, we aim to address the role of histone chaperone Hira in transcriptional regulation and chromatin configuration changes at molecular level and shed more light on the intrinsic mechanisms governing oocyte development. We are going to apply the recently developed single cell DNase 1 hypersensitivity sites detection assay to generate the first chromatin accessibility map in developing oocytes. Meanwhile, single oocyte RNA sequencing method will be used to generate gene regulatory network (GRN) in different stage oocytes. We will also combine dry (bioinformatics) and wet (experimental) lab approaches to identify and verify key players in the gene regulatory network. ..Oocyte is the only system that is able to fulfil complete epigenetic reprograming of somatic cell. Hence, the findings of this proposed project will help us to understand the underlying molecular mechanism of somatic cell reprogramming and enable us to design better, more efficient protocols and technologies for in vitro epigenetic reprogramming such as generation of induced pluripotent stem cells (iPSCs). Construction of reliable gene regulatory network in oocyte will help us to understand key determinants of the competent oocyte, which in turn makes it possible to select oocyte based on markers for assisted reproductive technologies (ART) and also provide candidate biomarkers for early diagnosis of human reproductive diseases such as premature ovarian failure (POF).

卵母细胞发育是一个复杂而漫长的过程。在此期间，卵母细胞需要转录和储存大量的RNA为受精和胚胎发育做准备。卵母细胞特异性敲除组蛋白分子伴侣Hira导致卵巢萎缩和卵母细胞死亡，但分子机理尚不清楚。本研究将单细胞分析技术与高通量测序技术相结合，首次构建卵母细胞染色质可接近性图谱。并通过单卵子RNA测序技术，构建卵母细胞发育过程中基因调控网络。结合生物信息学分析和实验验证法着重研究Hira调控卵母细胞染色质可接近性和基因转录的分子机理，鉴别和验证卵母细胞发育过程中的关键调节因子，加深对卵母细胞发育这一基础生物学问题分子水平的理解。卵母细胞是唯一具备重编程体细胞基因组的细胞体系。因此，本研究的发现将有助于理解体细胞重编程的分子机理，有助于研发更高效的诱导干细胞建立方法，服务于再生医学。同时，基因调控网络的构建和关键调节因子的鉴别也为辅助生殖医学筛选“优质”卵子和诊断卵巢早衰等疾病提供候选分子标记。

哺乳动物卵子和早期胚胎发育受到多种因素的精细调控，然而其分子机理尚未完全阐明。本研究结合卵母细胞特异性基因敲除手段和高通量测序技术，获得了生长期和成熟期两个不同发育阶段小鼠卵母细胞DNase1-seq和RNA-seq数据。通过生物信息学分析发现Hira缺失导致卵母细胞染色质可及性增强，转录组类似于2-细胞转录组，尤其Zscan4等2-细胞特异性基因提前表达，表明染色质可及性直接影响基因表达。发现组蛋白变异体H3.3及其分子伴侣Hira对Major Satellite区域的转录和抑制具有重要的调控作用，可直接影响卵母细胞和早期胚胎发育潜能。此外，发现组蛋白分子伴侣Asf1a和Asf1b以及新型表观遗传修饰即组蛋白乳酸化在小鼠卵子发育和早期胚胎发育过程中具有重要调控作用。同时发现常用化疗药物5-FU在雌性小鼠引起可逆性生殖毒性。本项研究产出9篇学术论文，研究成果加深了哺乳动物卵子和早期胚胎发育的表观遗传调控机理，鉴别了调控卵子和早期胚胎发育的重要调节因子，对不孕不育等生殖相关疾病的诊断和治疗具有重要参考价值。