DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [RNA-seq]

DNA methylation serves a stable gene regulatory function in mature somatic cells. In the germ line and during early embryogenesis, however, DNA methylation undergoes global erasure and re-establishment to support germ cell and embryonic development. While de novo DNA methylation during male germ cell development is essential for setting genomic imprints, possible other intergenerational roles for paternal DNA methylation following fertilization are unknown. To address this question, we reduced the level of DNA methylation in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. Mutant male germ cells nevertheless completed their differentiation to sperm. We observed that DNMT3A serves a largely maintenance-like methylation function at many intragenic sites in undifferentiated spermatogonia while DNMT3B catalyzes de novo methylation during spermatogonial differentiation. In spermatogonia, the acquisition of DNA methylation and deposition of H3K4me3 occur mutually exclusive. Failing de novo DNA methylation in spermatogonia leads to increased nucleosome occupancy in mature sperm at sites with high CpG content, reinforcing the model that DNA methylation constrains nucleosome retention in sperm. To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a highly sensitive transposon-based tagging assay for modified chromatin. Our data show that reduced DNA methylation in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently from paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNA methylation directs chromatin formation during early embryonic development. To assess the role of these proteins during adult spermatogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the transcriptome status of control and mutant undifferentiated spermatogonia, differentiated spermatogonia and sperm by total RNA sequencing.DNA甲基化（DNA methylation）在成熟体细胞中行使稳定的基因调控功能。但在生殖系（germ line）及早期胚胎发生（early embryogenesis）过程中，DNA甲基化会经历全局性的擦除与重建，以保障生殖细胞与胚胎的正常发育。尽管雄性生殖细胞发育阶段的从头DNA甲基化（de novo DNA methylation）对建立基因组印记（genomic imprints）不可或缺，但受精后父本DNA甲基化是否存在其他代间调控功能仍有待阐明。为解答该科学问题，我们通过在未分化精原细胞（undifferentiated spermatogonia）中条件性敲除（conditional gene deletion）从头DNA甲基转移酶（de novo DNA methyltransferase）DNMT3A与DNMT3B，降低了发育中雄性生殖细胞的DNA甲基化水平。值得注意的是，突变体雄性生殖细胞仍可完成向精子的分化全过程。研究发现，DNMT3A在未分化精原细胞的多数基因内位点上主要发挥类似维持型甲基化的功能，而DNMT3B则在精原细胞（spermatogonia）分化阶段催化从头甲基化。在精原细胞中，DNA甲基化的获取与组蛋白H3赖氨酸4三甲基化（H3K4me3）的沉积呈互斥关系。精原细胞中从头DNA甲基化的缺失，会导致成熟精子高CpG位点的核小体占据率（nucleosome occupancy）升高，这进一步验证了DNA甲基化可限制精子内核小体滞留的模型。为探究精子染色质异常对胚胎染色质形成的影响，我们采用针对修饰染色质的高灵敏度转座子标记法（transposon-based tagging assay），对二细胞胚胎（2-cell embryos）中父本等位基因（paternal alleles）与母本等位基因（maternal alleles）的H3K4me3占据情况进行了检测。结果显示，精子中DNA甲基化水平降低会使父本等位基因在早期胚胎中更易建立H3K4me3修饰，且该过程不依赖于精子携带的H3K4me3的父本遗传。综上，本研究首次提供直接证据表明，父本遗传的DNA甲基化可调控早期胚胎发育过程中的染色质构建。为探究这两种蛋白在成年精子发生（spermatogenesis）中的功能，我们构建了条件性敲除模型：以在出生后未分化及分化精原细胞（differentiated spermatogonia）中具有活性的Stra8启动子（promoter）驱动改良型iCre重组酶（iCre recombinase）转基因，从而特异性切除Dnmt3a与Dnmt3b的flox等位基因（floxed alleles，即两侧带有loxP位点的等位基因）。随后我们通过总RNA测序（total RNA sequencing），分析了对照组与突变组的未分化精原细胞、分化精原细胞及精子的转录组状态。