Nucleosome preservation in mammalian sperm: an epigenetic program ensuring the healthy male reproduction

哺乳动物精子中的核小体保存：确保男性健康生殖的表观遗传程序

• 批准号: 270041755
• 负责人: Professorin Dr. Undraga Schagdarsurengin
• 金额: --
• 依托单位: Laboratorium für funktionale Genomanalyse (LAFUGA)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/270041755?language=en
• 关键词: Nucleosome; preservation; mammalian; sperm; epigenetic

Based on current scientific knowledge, the sperm epigenome, especially the nucleosomes that were excluded from protamine-exchange, may potentially direct certain processes in early development. Our previous work showed that sperm-specific nucleosomes are largely retained in distal intergenic regions and associated with centromere repeats and retrotransposons, particularly those known as long, interspersed nuclear elements-1 (LINE1s). Remarkably, promoters and introns of genes relevant for pre-implantation development (e.g., factors for RNA-processing, signal transduction, and mitochondrial function) are also nucleosome-enriched. We reasoned that aberrations with regard to nucleosome-preservation pattern and nucleosome-associated genomic loci in sperm might cause severe errors in post-fertilization processes, and hence, may lead to idiopathic male infertility. The proposed project will address some crucial, open questions regarding the function of sperm nucleosomes, mechanisms that promote their preservation, and the potential risk of nucleosome aberrations for male fertility. Accordingly, our aims are: 1) To analyze the epigenetic status (CpG-methylation, histone modifications) of previously identified nucleosome-enriched gene promoters in a considerable number of sperm samples from fertile men. This point is essential to determine whether epigenetic variability in nucleosome-preserved promoters exists in the fertile population, and hence, can be tolerated; 2) To analyze the mRNA expression of human and bovine orthologous genes with nucleosome-enriched promoters, in early bovine embryos. This point is essential in selecting evolutionarily conserved genes, which are paternally-premarked and are relevant for mammalian pre-implantation development; 3) To analyze the promoters of the candidate genes selected in Aim 2 to determine their epigenetic status in sperm from sub-fertile patients; 4) To identify genome-wide aberrations related to nucleosome-preservation patterns in sperm. The mono-nucleosomal DNA from sub-fertile patients will be deep sequenced and compared to our previous data generated in fertile men. Moreover, the sperm methylome will be compared between sperm samples from sub-fertile and fertile men; 5) To elucidate the mechanisms that allow LINE1s to retain nucleosomes. We will analyze LINE1 mRNA expression and LINE1 CpG-methylation in different stages of spermatogenesis. LINE1 methylation will be compared to CpG-methylation in other repetitive DNA elements; 6) Our preliminary study showed that nucleosome-to-protamine exchange in spermiogenesis was accompanied by DNA hydroxymethylation. To elucidate the link between DNA demethylation and nucleosome preservation, we will analyze the hydroxymethylomes in spermatocytes and spermatids.

根据目前的科学知识，精子表观基因组，特别是被排除在鱼精蛋白交换之外的核小体，可能在早期发育过程中潜在地指导某些过程。我们以前的工作表明，精子特异的核小体主要保留在远端的基因间隔区，并与着丝粒重复和反转录转座子有关，特别是那些被称为长穿插核素-1(LINE1s)的核小体。值得注意的是，与植入前发育相关的基因的启动子和内含子(例如，RNA处理、信号转导和线粒体功能的因子)也富含核小体。我们推测，精子中核小体保存模式和核小体相关基因组座位的异常可能会导致受精后过程中的严重错误，从而可能导致特发性男性不育。拟议的项目将解决一些关键的开放问题，涉及精子核小体的功能，促进其保存的机制，以及核小体异常对男性生育能力的潜在风险。因此，我们的目标是：1)分析在相当数量的生育男性精子样本中先前发现的核小体富集型基因启动子的表观遗传学状态(CpG甲基化、组蛋白修饰)。这一点对于确定核小体保存的启动子的表观遗传变异是否存在于可育群体中并因此是可以容忍的是至关重要的；2)分析人和牛的同源基因在牛早期胚胎中的mRNA表达。这一点对于选择进化上保守的基因是至关重要的，这些基因是父系预先标记的，与哺乳动物着床前的发育相关；3)分析Aim 2中选择的候选基因的启动子，以确定它们在亚生育患者精子中的表观遗传学状态；4)识别与精子中核小体保存模式相关的全基因组异常。来自亚生育患者的单核小体DNA将被深度测序，并与我们之前在生育男性中产生的数据进行比较。此外，还将比较亚生育男性和生育男性的精子样本中的精子甲基组；5)阐明LINE1保留核小体的机制。我们将分析LINE1mRNA在精子发生的不同阶段的表达和LINE1CpG甲基化。6)我们的初步研究表明，精子发生过程中的核小体到鱼精蛋白的交换伴随着DNA羟甲基化。为了阐明DNA去甲基化与核小体保存之间的联系，我们将分析精母细胞和精子细胞中的羟甲基体。