Mechanisms and function of de novo DNA methylation in placental development [RNA-Seq]

DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We demonstrate that each of the DNMTs is required to establish the placental methylome and is distinctly targeted to genome based on underlying chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired development of the placental maternal-foetal interface. Critically, loss of DNA methylation in the placenta did not lead to abnormalities in lineage specification or cell identity, but to defective formation and vascularisation of the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental trophoblast cells, we were able to rescue the placental phenotype and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that the principal function of DNA methylation during embryogenesis is to regulate placental function, which in turn is critical for embryo survival. Using low-input RNA-seq and post bisulphite adaptor tagging (PBAT), we then assayed gene expression and genome-wide DNA methylation in E7.5 ExE and epiblast from Dnmt3a KO, Dnmt3b KO, Dnmt3a/b double KO (DKO), Dnmt3l KO and wildtype (WT) controls. The de novo DNMT enzymes have been shown to interact with modified histone tails, which modulates their catalytic activity and genome localisation in vitro. Thus, we generated ultra-low input ChIP-seq data for H3K4me1 and H3K27ac to combine with published datasets for H3K4me3, H3K27me3 and H3K36me3 in E6.5 ExE, immediately preceding the completion of de novo DNA methylation. To investigate the placental phenotype of Dnmt3b KO at the molecular level, we generated single nuclei RNA sequencing data from E12.5 placentas from a Dnmt3b KO and WT control.DNA甲基化是一类具有抑制作用的表观遗传修饰，对生物体发育不可或缺；缺乏从头DNA甲基转移酶（de novo DNA methyltransferases, DNMTs）的小鼠会出现胚胎及围产期致死表型，这便是该修饰重要性的典型例证。本研究系统解析了DNMT3A、DNMT3B与DNMT3L在小鼠胎盘基因调控及发育过程中的功能。我们证实，所有上述DNA甲基转移酶均参与建立胎盘甲基化组，且会依据基因组固有染色质特征被特异性靶向至不同区域。敲除Dnmt3b会导致滋养层细胞系中生殖系基因的去抑制，同时破坏胎盘母胎界面的正常发育。尤为关键的是，胎盘中DNA甲基化的缺失并未引发细胞谱系特化或细胞身份的异常，而是造成胎盘迷路层的形成与血管生成缺陷。我们利用Sox2-Cre系统在胚胎中敲除Dnmt3b，同时保留胎盘滋养层细胞中的Dnmt3b表达，成功挽救了胎盘表型，进而逆转了胚胎致死性——此时Dnmt3b缺陷型胚胎可存活至出生。综上，胚胎发生过程中DNA甲基化的核心功能是调控胎盘功能，而胎盘功能正常对于胚胎存活至关重要。我们采用低起始量RNA测序（RNA sequencing, RNA-seq）与亚硫酸氢盐衔接子标记测序（post bisulphite adaptor tagging, PBAT），对E7.5时期的胚外外胚层（extraembryonic ectoderm, ExE）与上胚层开展了基因表达与全基因组DNA甲基化分析，所检测的样本包括Dnmt3a敲除（knockout, KO）、Dnmt3b敲除、Dnmt3a/b双敲除（double knockout, DKO）、Dnmt3l敲除及野生型（wildtype, WT）对照。已有研究显示，从头DNA甲基转移酶可与修饰后的组蛋白尾段相互结合，这一相互作用会在体外调控其催化活性与基因组定位。据此，我们针对H3K4me1与H3K27ac生成了超低起始量染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）数据，并将其与已发表的E6.5胚外外胚层中H3K4me3、H3K27me3及H3K36me3的测序数据集进行整合分析——该时期恰处于从头DNA甲基化完成的前夜。为从分子层面深入探究Dnmt3b敲除小鼠的胎盘表型，我们从Dnmt3b敲除与野生型对照的E12.5胎盘中获取了单细胞核RNA测序数据。