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. Overall design: 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）在成熟体细胞中发挥稳定的基因调控功能。然而在生殖细胞系以及早期胚胎发生过程中，DNA甲基化会经历全局性的擦除与重建，以支持生殖细胞与胚胎发育。尽管雄性生殖细胞发育过程中的从头DNA甲基化（de novo DNA methylation）对于建立基因组印记至关重要，但受精后父本DNA甲基化是否存在其他代际功能仍未明确。
为解答这一问题，我们通过在未分化精原细胞中条件性敲除从头DNA甲基转移酶DNMT3A与DNMT3B，下调了发育中雄性生殖细胞内的DNA甲基化水平。尽管如此，突变型雄性生殖细胞仍可完成向精子的分化。我们发现，DNMT3A在未分化精原细胞的众多基因内位点上主要发挥维持型甲基化功能，而DNMT3B则在精原细胞分化过程中催化从头甲基化。
在精原细胞中，DNA甲基化的获得与组蛋白H3赖氨酸4三甲基化（H3K4me3）的沉积呈现相互排斥的关系。精原细胞中从头DNA甲基化的缺失，会导致成熟精子中高CpG含量位点的核小体占据率升高，进一步支持了DNA甲基化可限制精子中核小体滞留的模型。
为探究精子染色质改变对胚胎染色质形成的影响，我们采用一种针对修饰染色质的高灵敏度转座子标记检测法（transposon-based tagging assay），对2细胞胚胎中父本与母本等位基因上的H3K4me3占据情况进行了检测。我们的数据显示，精子中DNA甲基化水平的下调会使父本等位基因在早期胚胎中易于建立H3K4me3，这一过程并不依赖于精子携带的H3K4me3的父本遗传。
综上，本研究首次提供证据表明，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形成。

实验整体设计：为探究这些蛋白质在成年精子发生过程中的功能，我们构建了条件性敲除模型：在该模型中，由受Stra8启动子调控的改进型iCre重组酶转基因驱动，实现Dnmt3a与Dnmt3b的flox等位基因的切除；该Stra8启动子在出生后未分化及分化型精原细胞中均具有活性。我们通过总RNA测序，检测了对照组与突变组的未分化精原细胞、分化型精原细胞以及精子的转录组状态。