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，特别是从鱼精蛋白交换中排除的核小体，可能潜在地指导早期发育的某些过程。我们以前的工作表明，精子特异性核小体主要保留在远端基因间区域，并与着丝粒重复序列和反转录转座子，特别是那些被称为长，散置核元件-1（LINE 1）。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加工、信号转导和线粒体功能的因子）也是核小体富集的。我们推测精子中核小体保存模式和核小体相关基因组位点的异常可能导致受精后过程的严重错误，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，开放的问题，关于精子核小体的功能，促进其保存的机制，以及核小体畸变对男性生育力的潜在风险。因此，我们的目标是：1）分析先前鉴定的核小体富集基因启动子在相当数量的生育男性精子样品中的表观遗传状态（CpG-甲基化、组蛋白修饰）。这一点是必不可少的，以确定核小体保存的启动子中是否存在表观遗传变异性在可育群体中，因此，可以容忍的; 2）分析的mRNA表达的人和牛的orthopathic基因与核小体富集的启动子，在早期牛胚胎。这一点是至关重要的，在选择进化上保守的基因，这是父系预先标记的，并与哺乳动物植入前发育; 3）分析的候选基因的启动子在目的2中选择，以确定其在精子的表观遗传状态从低生育力患者; 4）确定全基因组的精子核小体保存模式相关的畸变。将对来自低生育力患者的单核小体DNA进行深度测序，并与我们先前在生育力男性中产生的数据进行比较。此外，精子甲基化组将在来自亚生育力和生育力男性的精子样品之间进行比较; 5）阐明允许LINE 1保留核小体的机制。我们将分析LINE 1 mRNA表达和LINE 1 CpG甲基化在精子发生的不同阶段。LINE 1甲基化将与其他重复DNA元件中的CpG甲基化进行比较; 6）我们的初步研究表明，精子发生中的核小体-鱼精蛋白交换伴随着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子细胞中的羟甲基化组。

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.