Human Sperm Epigenomes and Transcriptomes Reveal Novel Features of Enhancers, Sex Chromosomes, piRNAs, Gametogenesis, and Inherited Small RNAs (Bisulfite-Seq). Homo sapiens

To better understand the paternally inherited epigenome and transcriptome, we profiled multiple histone modifications, histone H3.3 variant, cytosine DNA methylation at base-pair resolution, and small RNA transcriptomes. We report new and surprising features of enhancers, pluripotency factor targets, spermiogenesis genes, piRNA clusters, CpG islands, partially methylated domains, imprinted loci, repetitive elements and sex chromosomes. First, we find that enhancers for embryonic transcription factors bear H3K27me3, but low H3K14ac and DNA methylation -- consistent with poising for future activation -- whereas enhancers near genes active in gametogenesis have high H3K27ac/H3K14ac, consistent with past activation. Second, binding site regions (in ES cells) for the pluripotency factors SOX2 and NANOG are DNA methylated in sperm, suggesting a need for zygotic demethylation/reprogramming. Unexpectedly, genes for spermiogenesis (but not spermatogenesis), and almost half of piRNA cluster promoters, bear both H3K4me3 and DNA methylation, a co-incidence not previously observed in mammals. Furthermore, we find major assymetries in H3.3 and H3K9me3 density between the X and Y chromosomes after MSCI. Lastly, deep sequencing reveals >10-fold increase in the known repertoire of retained/inherited miRNAs and piRNAs than previous estimates. Thus, the paternal epigenome contributes a mosaic of epigenetic regions that reflect past gametogenesis or poising for subsequent embryonic developmental regulation -- and a complex repertoire of small RNAs. Overall design: Sperm from two fertile donors were subjected independently to genome-wide bisulfite sequencing.

为深入解析父本遗传的表观基因组与转录组，我们以碱基对分辨率对多种组蛋白修饰、组蛋白H3.3变体、胞嘧啶DNA甲基化及小RNA转录组开展了全景式表征分析。本研究报道了增强子、多能性因子靶标、精子形成相关基因、piRNA（Piwi-interacting RNA）簇、CpG岛（CpG island）、部分甲基化区域、印记基因座、重复序列元件与性染色体的全新且颇具颠覆性的特征。其一，胚胎转录因子的增强子携带H3K27me3修饰，但H3K14ac与DNA甲基化水平较低——这与其处于未来激活前的预激活筹备状态相符；而配子发生相关基因附近的增强子则呈现高H3K27ac与H3K14ac修饰水平，这与其已完成激活的状态一致。其二，胚胎干细胞（ES细胞）中多能性因子SOX2与NANOG的结合区域在精子中存在DNA甲基化修饰，这提示合子阶段需要进行去甲基化/重编程过程。出人意料的是，精子形成相关基因（而非精子发生相关基因），以及近半数piRNA簇启动子，同时携带H3K4me3与DNA甲基化修饰——这种共修饰现象此前在哺乳动物中从未被观测到。此外，我们发现经过减数分裂性染色体失活（MSCI）后，X与Y染色体之间在H3.3与H3K9me3的富集密度上存在显著不对称性。最后，深度测序结果表明，保留/遗传的miRNA与piRNA的已知库规模较此前预估提升了10倍以上。综上，父本表观基因组包含一系列表观遗传区域，这些区域既反映了过往的配子发生过程，也为后续胚胎发育调控提供了预激活筹备状态，同时还携带一套复杂的小RNA库。整体实验设计：来自两名健康可育供体的精子样本分别独立开展了全基因组亚硫酸氢盐测序（bisulfite sequencing）。