Whole genome bisulfite sequencing of adult opossum tissues

Decades of work in placental (eutherian) species have constructed a paradigm of mammalian development, wherein the genome-wide erasure of parental DNA methylation is required for embryogenesis1-10. Whether such DNA methylation reprogramming is, in fact, conserved in other mammals is unknown. To resolve this point, we generated base-resolution DNA methylation maps in gametes, embryos and adult tissues of the opossum marsupial Monodelphis domestica, revealing extensive variations from the eutherian-derived model. In stark contrast with eutherians, the marsupial genome remains hypermethylated during the cleavage stages and in the embryo proper of the blastocyst. In the extra-embryonic trophectoderm DNA methylation is reduced, suggesting an important evolutionary conserved function for DNA hypomethylation in formation of the mammalian placenta. Furthermore, unlike in eutherians, the inactive X chromosome becomes globally DNA hypomethylated during embryogenesis. Using our DNA methylation profiles, we identify a candidate mechanism for imprinted X-inactivation in marsupials, via maternal promoter DNA methylation of the Xist-like non-coding RNA RSX11. How mammalian embryos employ DNA methylation to regulate their development is therefore more mechanistically diverse than current models can accommodate. From each animal the same three tissues were extracted: brain, liver, spleen. There are six animals, three female and three male, so 18 samples in total. All tissue was snap frozen in liquid nitrogen and stored at -80°C until library preparation.

针对有胎类（真哺乳亚纲，eutherian）物种的数十年研究已构建出哺乳动物发育的经典范式，即全基因组范围内的亲本DNA甲基化擦除是胚胎发生所必需的过程1-10。目前尚不清楚这类DNA甲基化重编程是否在其他哺乳动物类群中保守存在。为厘清这一问题，我们对有袋动物灰短尾负鼠（Monodelphis domestica）的配子（gametes）、胚胎及成体组织绘制了碱基分辨率（base-resolution）的DNA甲基化图谱，结果显示其与基于真哺乳亚纲的经典模型存在广泛差异。与真哺乳亚纲动物截然不同的是，有袋动物的基因组在卵裂阶段以及囊胚的胚胎本体区域中仍维持高甲基化状态；而在胚外滋养外胚层（extra-embryonic trophectoderm）中，DNA甲基化水平有所降低，这提示DNA低甲基化在哺乳动物胎盘形成中具有进化保守的重要功能。此外，与真哺乳亚纲动物不同，有袋动物的失活X染色体（inactive X chromosome）在胚胎发生过程中会发生全基因组范围的DNA低甲基化。借助我们绘制的DNA甲基化图谱，我们鉴定出有袋动物印记X失活（imprinted X-inactivation）的候选机制：即通过Xist样非编码RNA（non-coding RNA）RSX11的母源启动子（promoter）DNA甲基化实现。因此，哺乳动物胚胎利用DNA甲基化调控发育的分子机制，其多样性远超现有模型的认知范畴。本次实验中，每只受试动物均采集相同的三种组织：大脑、肝脏与脾脏。共使用6只动物，其中雌性3只、雄性3只，总计18个样本。所有组织均经液氮快速冷冻后，于-80℃条件下保存直至文库构建（library preparation）。