GGNBP2介导组蛋白泛素化修饰调控Hox基因转录在精子发生中的功能研究

Spermatogenesis includes spermatogonial proliferation and differentiation, spermatocyte meiosis and spermiogenesis. This process is regulated by many genes, and Hox gene as the main control gene of cell proliferation and differentiation plays an important role in spermatogenesis. Our previous research revealed that gametogenetin binding protein Ggnbp2-/- mice showed azoospermic phenotype, lower amount of spermatids in each stage of the seminiferous tubules, and dramatic morphological defects of developing spermatids including irregularly shaped acrosomes and nucleolus and deformity as well as compromised DNA double strand damage repair mechanism. In addition, we also showed that GGNBP2 could interact with ASXL1 and ubiquitin binding enzyme E2B to affect histone ubiquitination. However, the mechanism is still unclear. In this research, we studied the biological function of GGNBP2 protein and the mechanism of histone ubiquitination in vitro and in vivo, and revealed the mechanism of the protein regulating Hox gene transcription using luciferase assay and chromatin immunoprecipitation (ChIP). Besides, The effect of target Hox genes on the differentiation of mouse spermatocyte was verified in cell model. This study will enrich the mechanism of epigenetic modification of spermatogenesis and provide experimental and theoretical basis for the screening of male non-obstructive azoospermia in clinical work.

精子发生包括精原细胞增殖分化、精母细胞减数分裂和精子细胞成熟变形，该过程受多种基因调控，其中Hox基因作为细胞增殖和分化的主控基因发挥重要作用。申请人已研究证实配子生成素结合蛋白Ggnbp2-/-小鼠无精子症表型，睾丸组织各级生精小管中各期精子细胞数量下降，精子细胞顶体畸形，精母细胞DNA双链断裂修复机制障碍。此外前期结果发现GGNBP2分别能与多梳蛋白ASXL1和泛素结合酶E2B相互作用，影响睾丸生精细胞组蛋白泛素化修饰，但修饰机制尚不清楚。本项目从体内外实验研究GGNBP2蛋白参与组蛋白泛素化修饰机制，从而揭示其生物学功能，同时采用荧光素酶报告系统和染色质免疫共沉淀技术(ChIP)探讨其调控Hox基因转录模式，并在细胞模型上验证Hox靶基因影响小鼠精母细胞分化功能，该研究将丰富精子发生表观遗传学修饰机制，为今后临床工作中男性非梗阻性无精子症的筛查提供实验依据和理论基础。

我们前期证实配子生成素结合蛋白Ggnbp2-/-小鼠无精子症表型，发现该基因敲除影响睾丸生精细胞组蛋白泛素化修饰。本项目从体内外实验研究GGNBP2蛋白参与组蛋白泛素化修饰机制，发现GGNBP2可能通过调控去泛素化酶复合体ASXL1/BAP1结合，或者泛素特异性蛋白酶USP表达影响组蛋白H2AK119去泛素化。同时GGNBP2通过影响泛素结合酶E2B和泛素连接酶RNF40相互作用，调控组蛋白H2BK120泛素化水平。组蛋白泛素化和甲基化之间存在交联作用，GGNBP2可通过影响H3甲基化酶活性，影响组蛋白H3甲基化修饰。在精子发生减数分裂和DNA损伤修复机制、以及稳定染色质核小体中发挥重要作用。此外我们在Ggnbp2敲除小鼠睾丸组织发现了一个睾丸特异性表达的长链非编码RNA(lncRNA-Slfn5os)。Slfn5os的表达水平对Slfn5的mRNA表达具有负调节作用，该基因过表达可以通过影响类固醇生成基因和蛋白的表达，影响睾酮的产生，本项目初步揭示了其影响睾酮生成的分子机制，为今后性腺功能减退及雄性发育的研究提供了研究方向。