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

根据目前的科学知识，精子表观基因组，特别是排除在蛋白蛋白交换之外的核小体，可能潜在地指导早期发育的某些过程。我们之前的工作表明，精子特异性核小体大部分保留在远端基因间区域，并与着丝粒重复和反转录转座子相关，特别是那些被称为长，分散核元件-1 （LINE1s）的核小体。值得注意的是，与着床前发育相关的基因启动子和内含子（例如，rna加工、信号转导和线粒体功能的因子）也富含核小体。我们推断，精子中核小体保存模式和核小体相关基因组位点的畸变可能导致受精后过程中的严重错误，因此可能导致特发性男性不育。拟议的项目将解决一些关键的、悬而未决的问题，包括精子核小体的功能、促进核小体保存的机制，以及核小体畸变对男性生育能力的潜在风险。因此，我们的目标是：1)分析大量可育男性精子样本中先前鉴定的核小体富集基因启动子的表观遗传状态（cpg甲基化，组蛋白修饰）。这一点对于确定核小体保存启动子的表观遗传变异性是否存在于可育群体中，并因此可以被容忍是至关重要的；2)分析人类和牛同源基因富集核小体启动子mRNA在早期牛胚胎中的表达。这一点对于选择进化保守的基因至关重要，这些基因是父亲预先标记的，与哺乳动物着床前发育有关；3)分析Aim 2中选择的候选基因的启动子，确定其在亚生育患者精子中的表观遗传状态；4)鉴定与精子核小体保存模式相关的全基因组畸变。来自低生育能力患者的单核体DNA将被深度测序，并与我们之前在有生育能力的男性中产生的数据进行比较。此外，将比较亚生育能力和生育能力男性的精子样本之间的精子甲基组；5)阐明允许line1保留核小体的机制。我们将分析LINE1 mRNA的表达和LINE1 cpg -甲基化在精子发生的不同阶段。LINE1甲基化将与其他重复DNA元件的cpg甲基化进行比较；6)我们的初步研究表明，精子发生过程中核小体与鱼精蛋白的交换伴随着DNA羟甲基化。为了阐明DNA去甲基化和核小体保存之间的联系，我们将分析精母细胞和精母细胞中的羟甲基组。

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.