Density and methylation state of CpG dinucleotides define histone variant specific retention of nucleosomes in mouse spermatozoa

Nucleosomes are the principal packaging units of chromatin and critical for gene regulation and genome stability. In mammals, a subset of nucleosomes fail to be replaced by protamines during spermatogenesis and are retained in mature spermatozoa providing opportunities for paternal epigenetic transmission. In humans, the remaining 10% localize at regulatory elements of genes. To assess evolutionary conservation and to dissect the molecular logic underlying nucleosome retention, we determined the genome wide nucleosome occupancy in mouse spermatozoa that only contain 1% residual histones. In striking contrast to mammalian somatic cells and haploid round spermatids, we observe high enrichment of nucleosomes at CpG-rich sequences throughout the genome, at conserved regulatory sequences as well as at intra- and intergenic regions and repetitive DNA. This preferred occupancy occurs mutually exclusive with DNA methylation both in mouse and human sperm. At unmethylated CpG-rich sequences, residing nucleosomes are largely composed of the H3.3 histone variant, and trimethylated at lysine 4 (H3K4me3). Both canonical H3.1/H3.2 and H3.3 variant histones are present at promoters marked by Polycomb-mediated H3K27me3, which is strongly predictive for gene repression in pre-implantation embryos. Our data indicate important roles of DNA sequence composition, DNA methylation, variant H3.3 and canonical H3.1/H3.2 histones and associated modifications in nucleosome retention versus eviction during the histone-to-protamine remodeling process in elongating spermatids and potentially in epigenetic inheritance by nucleosomes between generations. Identification of histone, histone variant and histone modification states in round spermatids and sperm核小体（nucleosome）是染色质的主要包装单元，对基因调控与基因组稳定性至关重要。在哺乳动物中，部分核小体无法在精子发生过程中被鱼精蛋白替换，而是保留于成熟精子内，为父代表观遗传传递创造了条件。在人类中，剩余10%的核小体定位于基因的调控元件区域。 为评估进化保守性并解析核小体保留的分子机制，我们对仅含有1%残留组蛋白的小鼠精子开展了全基因组核小体占据谱分析。与哺乳动物体细胞及单倍体圆形精子细胞形成鲜明对比的是，我们在小鼠精子中观察到，核小体在全基因组范围内的CpG富集序列、保守调控序列、基因内与基因间区域以及重复DNA区域均呈现高度富集。这种偏好性占据模式在小鼠与人类精子中均与DNA甲基化呈互斥关系。 在未甲基化的CpG富集序列区域，驻留的核小体主要由组蛋白变体H3.3构成，且其赖氨酸4位点发生三甲基化修饰（H3K4me3）。在由多梳蛋白（Polycomb）介导的H3K27me3修饰标记的启动子区域，同时存在经典组蛋白H3.1/H3.2与组蛋白变体H3.3；该修饰类型可强烈预示着床前胚胎中的基因阻遏过程。 我们的研究数据表明，在伸长型精子细胞的组蛋白-鱼精蛋白重塑过程中，DNA序列组成、DNA甲基化、组蛋白变体H3.3与经典组蛋白H3.1/H3.2及其相关修饰在核小体保留与移除环节中发挥着重要作用，且该过程可能通过核小体参与代际间的表观遗传传递。本研究对圆形精子细胞与精子中的组蛋白、组蛋白变体及组蛋白修饰状态进行了鉴定。