Deciphering the role of sex steroids in male germline development and epigenetic reprogramming.

围产期发育对男性生殖细胞编程至关重要。在这个阶段，表观遗传重编程发生，包括基因组几乎完全的DNA去甲基化，随后是部分由组蛋白修饰和转录因子结合引导的渐进从头DNA甲基化(DANme)。这建立了精子表观基因组，在一定程度上指导胚胎发育。重要的是，表观遗传重编程容易在围产期暴露于内分泌干扰物(EDCs)，导致细胞功能异常和跨代遗传。然而，我们不知道所涉及的机制，这限制了我们补救这些负面影响的能力。我的研究项目旨在更好地了解哺乳动物围产期雄性生殖细胞发育的潜在分子基础，并阐明它如何受到内分泌细胞的错误调控。为此，我们建立了一种分离发育关键阶段的大鼠生殖细胞的方法，并在最近描述了DNAME的动态，六个组蛋白翻译后修饰(PTM)和表观遗传修饰的表达。由于内分泌细胞经典的作用是通过性类固醇信号传递，我们现在质疑性类固醇受体在表观遗传重新编程中的作用，这可能会低估这些污染物如何影响早期细胞编程并产生长期影响。为了解决这个问题，将追求三个具体目标。首先，我们的目标是确定性类固醇受体和从头开始的DNAME之间的联系。为此，我们将建立性类固醇受体在纯化生殖细胞整个发育过程中的动态表达，并通过蛋白质组学分析来表征它们的辅助因素。为了预测对新的5mC的影响，我们将定位性类固醇结合部位，并将它们与我们建立的DNAME发展图进行比较。其次，我们将测试妊娠期性激素失衡对男性生殖系表观基因组的影响。在表观遗传重编程期间，男性生殖细胞在宫内暴露于药物激动剂和性类固醇拮抗剂后将被纯化。利用测序数据，我们将确定对全基因组DNAME、特异组蛋白PTMS结合位点和男性生殖系基因表达的影响。第三，我们的目的是通过在发育中的雄性生殖细胞(体内和体外ESR1或Esr2基因在原代培养和永生化生殖细胞系中失活)建立DNAME和特异组蛋白PTMS结合位点的全基因组动力学来表征雌激素信号对雄性生殖系表观基因组的影响。围产期发育是细胞编程的关键时期，因为细胞终生获取信息。这项研究计划不仅将提高对雄性生殖细胞编程的基本理解，而且还将确定早期暴露于EDCs的影响。

Perinatal development is critical for male germ cells programming. At this stage, epigenetic reprogramming occurs, which consists of almost complete DNA demethylation of the genome followed by progressive de novo DNA methylation (DANme) guided in part by histone modifications and binding of transcription factors. This establishes the sperm epigenome that, in part, guides embryo development. Importantly, the epigenetic reprogramming is susceptible to perinatal exposure to endocrine disrupting chemicals (EDCs), leading to abnormal cellular function, and transgenerational inheritance. However, we do not know the mechanisms involved, which limits our ability to remedy these negative effects. My research program aims to better understand the underlying molecular basis for perinatal male germ cell development in mammals and elucidate how it can be misregulated by EDCs. For this purpose, we have established a method for sorting rat germ cells at key stages of development and have recently described the dynamics of DNAme, six histone post-translational modifications (PTMs) and expression of epigenetic modifiers. As EDCs classically act through sex steroid signalling, we now question the role of sex steroid receptors on the epigenetic reprogramming that could underly how these contaminants affect early cellular programming and have long term impact. To address this question, three specific aims will be pursued. First, we aim to determine the link between sex steroid receptors and de novo DNAme. To that end, we will establish the dynamic expression of sex steroid receptors in purified germ cells throughout development and characterize their co-factors by proteomic analysis. To predict influence on de novo 5mC, we will locate sex steroid binding sites and compare them to the developmental map of DNAme we have established. Second, we will test the impact of a sex steroid imbalance during gestation on the male germline epigenome. Male germ cells will be purified after in-utero exposure to pharmaceutical agonist and antagonist of sex steroid, during epigenetic reprogramming. Using sequencing data, we will determine the impact on genome wide DNAme, specific histone PTMs binding sites and gene expression in the male germline. Thirdly, we aim to characterize the influence of the estrogen signaling on the male germline epigenome by establishing the genome wide dynamics of DNAme and specific histone PTMs binding sites in developing male germ cells in which the Esr1 or Esr2 genes have been inactivated in vivo and in vitro in primary culture and an immortalized germ cell line. Perinatal development represents a key period in cellular programming, as cells acquire information for a lifetime. This research program will not only improve the fundamental understanding of male germ cell programming, but also identify the influence of early exposure to EDCs.