Genome-wide roles of DNA methyltransferases in mouse embryos

Mouse embryos acquire global DNA methylation of their genome during implantation. However the exact roles of DNA methyltransferases (DNMTs) in embryogenesis have not been studied comprehensively. Here we systematically analyze the consequences of genetic inactivation of Dnmt1, Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos and fibroblasts. We mapped DNA methylation by Whole Genome Bisulfite Sequencing (WGBS) in two independent WT, Dnmt1-/- and Dnmt3a-/- Dnmt3b-/- (DKO) E8.5 embryos. We also sequenced RRBS libraries from three Dnmt1-/- (D1KO_Rep1-3) and WT (D1WT_Rep1-3) littermate E8.5 embryos, as well as three Dnmt3a-/- Dnmt3b-/- (DKO_Rep1-3) together with two WT (D3abWT_Rep1-2), three Dnmt3a-/+ (D3aHet_Rep1-3), three Dnmt3a-/+ Dnmt3b-/+ (D3abHet_Rep1-3) littermate E8.5 embryos. To study the role of DNMT3A/B and DNMT1 in maintenance methylation, we generated Dnmt3aL2/L2; Dnmt3bL2/L2; CreERT2 and Dnmt1L2/L2; CreERT2 immortalized mouse embryonic fibroblasts (MEFs). The CreERT2 recombinase is activated by tamoxifen treatment to generate Dnmt3a Dnmt3b double conditional knockout (cDKO) and Dnmt1 conditional knockout (D1cKO) MEFs. We performed three independent tamoxifen induction experiments and profiled DNA methylation by RRBS in cells treated with Tamoxifen (Tam_Rep1-3) or not treated with Tamoxifen (noTam_Rep1-3) at various time points of culture (day 23 and day 69 for cDKO MEFs, day 5 and day 7 for D1cKO MEFs). The transcriptome of Dnmt mutant embryos was analyzed by RNA-seq. We sequenced RNA-seq libraries from three Dnmt1-/- (D1KO_Rep1-3) and three WT (D1WT_Rep1-3) littermate E8.5 embryos, as well as six Dnmt3a-/- Dnmt3b-/- E8.5 embryos (DKO_Rep1-6) together with two WT (D3abWT_Rep1-2) and four Dnmt3a-/+ (D3aHet_Rep1-4) littermate controls.小鼠胚胎在着床过程中会获得全基因组范围的DNA甲基化修饰。然而，DNA甲基转移酶（DNA methyltransferases, DNMTs）在胚胎发生过程中的具体功能尚未得到全面研究。本研究系统性分析了Dnmt1、Dnmt3a与Dnmt3b基因失活对小鼠胚胎及成纤维细胞的甲基化组与转录组的影响。我们通过全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing, WGBS），对两份独立的野生型（wild type, WT）、Dnmt1-/-以及Dnmt3a-/- Dnmt3b-/-（double knockout, DKO）E8.5小鼠胚胎进行了DNA甲基化图谱绘制。我们还对三份Dnmt1-/-（D1KO_Rep1-3）及野生型（D1WT_Rep1-3）同窝E8.5胚胎的简化基因组亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）文库进行了测序；此外还对三份Dnmt3a-/- Dnmt3b-/-（DKO_Rep1-3）胚胎、两份野生型（D3abWT_Rep1-2）胚胎、三份Dnmt3a-/+（D3aHet_Rep1-3）胚胎以及三份Dnmt3a-/+ Dnmt3b-/+（D3abHet_Rep1-3）同窝E8.5胚胎进行了测序。为研究DNMT3A/B与DNMT1在维持性甲基化中的功能，我们构建了携带Dnmt3aL2/L2; Dnmt3bL2/L2; CreERT2以及Dnmt1L2/L2; CreERT2的永生化小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）。通过他莫昔芬处理激活CreERT2重组酶，可分别获得Dnmt3a Dnmt3b双条件性敲除（cDKO）与Dnmt1条件性敲除（D1cKO）的MEFs。我们开展了三次独立的他莫昔芬诱导实验，并通过RRBS对不同培养时间点的细胞进行了DNA甲基化图谱分析：其中cDKO MEFs的采样时间为培养第23天与第69天，D1cKO MEFs的采样时间为培养第5天与第7天；处理组为他莫昔芬处理的细胞（Tam_Rep1-3），对照组为未处理的细胞（noTam_Rep1-3）。我们通过RNA测序（RNA sequencing, RNA-seq）分析了Dnmt突变胚胎的转录组：对三份Dnmt1-/-（D1KO_Rep1-3）及三份野生型（D1WT_Rep1-3）同窝E8.5胚胎，以及六份Dnmt3a-/- Dnmt3b-/- E8.5胚胎（DKO_Rep1-6）、两份野生型（D3abWT_Rep1-2）胚胎与四份Dnmt3a-/+（D3aHet_Rep1-4）同窝对照胚胎的RNA-seq文库进行了测序。