Regulation of chromatin remodeling during spermiogenesis

 描述(申请人提供)：圆形精子细胞分化为精子，称为精子发生，是一个复杂的过程，涉及顶体、鞭毛的形成和细胞核的凝聚。在为凝聚做准备的过程中，精子细胞核经历了戏剧性的染色质重塑，包括全基因组范围的转录停止，核小体组织的瓦解，以及组蛋白向鱼精蛋白的转变。这一过程是雄性生殖系独有的。小鼠模型已经表明，核凝结缺陷会导致男性不育；然而，调控这一复杂的全基因组过程的机制还不太清楚。我们的工作假设是基因组组织者蛋白CTCF协调伴随精子细胞分化的染色质重塑事件。在进化上保守的11个锌指蛋白CCCTC结合因子(CTCF)仅在小鼠圆形和早期伸长的精子细胞中表达，这与转录关闭和组蛋白替换的开始相吻合。在这个试验性的可行性资助方案中，我们将检验以下假设：1)CTCF作为转录因子和精子细胞基因组的组织者，促进染色质重塑；2)CTCF对于完成精子发生和男性生育是必不可少的。我们将进行CHIP-SEQ来确定CTCF在圆形精子细胞中的全基因组占有率，并使用Cre-loxP技术产生CTCF条件基因敲除小鼠，以测试CTCF对精子发生和男性生育的需求。CTCF是这一角色的理想候选者，因为它是一种多功能DNA结合蛋白，具有多种作用，包括染色质组织者的作用。CTCF小鼠和雄性生殖细胞特异性克隆品系(Stra8-iCre)都是现成的。这项建议将首次探索CTCF在男性生育中的作用。在育龄组中，每6对夫妇中就有1对受到不孕症的影响，其中男性占50%。如果CTCF耗尽导致精子成熟停滞，本研究将为男性不育提供一种新的小鼠模型。考虑到CTCF在建立和/或维持表观遗传标记方面的作用，所提出的基因敲除小鼠可能是一个有用的模型，可以用来理解使用不完全发育的精子细胞或精子的辅助生殖技术所涉及的风险。因此，从男性生殖健康的角度来看，这些研究具有重要的意义。

 DESCRIPTION (provided by applicant): The differentiation of round spermatids into spermatozoa, known as spermiogenesis, is a complex process involving the formation of the acrosome, the flagellum, and condensation of the nucleus. In preparation for condensation, the spermatid nucleus undergoes dramatic chromatin remodeling including genome-wide cessation of transcription, dismantling of the nucleosomal organization, and histone to protamine transition. This process is unique to the male germ line. Mouse models have shown that defects in nuclear condensation lead to male infertility; however, the mechanisms regulating this complex genome-wide process are not well understood. Our working hypothesis is that the genome organizer protein CTCF coordinates the chromatin remodeling events accompanying spermatid differentiation. The evolutionarily conserved eleven zinc finger protein CCCTC-binding factor (CTCF) is exclusively expressed within the round and early elongating spermatids in mice, coinciding with transcriptional shutdown and the onset of histone replacement. In this pilot, feasibility grant proposal we will test the hypotheses that: 1) CTCF functions as a transcription factor as well as an organizer of spermatid genome to facilitate chromatin remodeling and 2) CTCF is essential for the completion of spermatogenesis and male fertility. We will perform ChIP-seq to determine genome-wide occupancy of CTCF in round spermatids and generate CTCF conditional knockout mice using the Cre-loxP technology to test the requirement of CTCF for spermatogenesis and male fertility. CTCF is an ideal candidate for this role because it is a multifunctional DNA binding protein with diverse roles including that of a chromatin organizer. Floxed CTCF mice as well as the male germ cell-specific cre-deleter strain (Stra8-iCre) are readily available. This proposal will explore the role of CTCF in male fertility fr the first time. Infertility affects 1 in 6 couples in the reproductive age group, with the male facor accounting for 50% of those cases. If depletion of CTCF causes sperm maturation arrest, this study will provide a novel mouse model for male infertility. Given the role that CTCF plays in establishing and / or maintaining epigenetic marks, the proposed knockout mouse may be a useful model to understand the risks involved in Assisted Reproductive Technology using incompletely developed spermatids or sperm. Thus, the studies are highly significant from the point of view of male reproductive health.