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

破译性类固醇在雄性种系发育和表观遗传重编程中的作用。

• 批准号: RGPIN-2022-05110
• 负责人: Delbes, Geraldine
• 金额: $ 2.4万
• 依托单位: Institut national de la recherche scientifique
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743849
• 关键词: Deciphering; role; sex; steroids; male

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.

围产期发育对男性生殖细胞编程至关重要。在这个阶段，发生表观遗传重编程，其包括基因组的几乎完全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性从头DNA甲基化（DANme）。这建立了精子表观基因组，部分指导胚胎发育。重要的是，表观遗传重编程易受围产期内分泌干扰物（EDCs）的影响，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，并阐明它如何被内分泌干扰物错误调节。为此，我们已经建立了一种方法来分选大鼠生殖细胞在关键阶段的发展，最近描述了动态的DNAme，六组蛋白翻译后修饰（PTM）和表观遗传修饰剂的表达。由于EDCs通常通过性类固醇信号传导起作用，我们现在质疑性类固醇受体在表观遗传重编程中的作用，这可能是这些污染物如何影响早期细胞编程并产生长期影响的基础。为解决这一问题，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DNA之间的联系。为此，我们将在整个发育过程中建立纯化生殖细胞中性类固醇受体的动态表达，并通过蛋白质组学分析表征其辅助因子。为了预测对从头5mC的影响，我们将定位性类固醇结合位点，并将其与我们已经建立的DNAme发育图进行比较。其次，我们将测试妊娠期间性类固醇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，雄性生殖细胞将在子宫内暴露于性类固醇的药物激动剂和拮抗剂后进行纯化。使用测序数据，我们将确定对全基因组DNAme、特定组蛋白PTM结合位点和雄性生殖系中基因表达的影响。第三，我们的目标是通过建立发育中的雄性生殖细胞中的DNAme和特异性组蛋白PTM结合位点的全基因组动力学来表征雌激素信号传导对雄性生殖系表观基因组的影响，其中Esr1或Esr2基因在原代培养物和永生化生殖细胞系中已经在体内和体外失活。 围产期发育是细胞编程的关键时期，因为细胞一生都在获取信息。这项研究计划不仅将提高对男性生殖细胞编程的基本理解，还将确定早期暴露于内分泌干扰物的影响。