DNA Methylation at N6-Adenine in Placental Trophoblast Development

滋养层细胞在胎儿和母亲之间的沟通中起着至关重要的作用。未能准确地建立滋养层谱系会导致胚泡阶段之前的发育停滞、着床失败、早期流产和胎盘异常。最近，我们首次在哺乳动物基因组中发现了N6-腺嘌呤(N6-mA)上的一种新的DNA甲基化(Wu等人，自然，2016；谢等人，Cell，2018)。我们最新的初步研究表明，N6-mA主要存在于早期胚胎的滋养外胚层/滋养层谱系中，并在小鼠和牛中保守，可能在所有哺乳动物中都存在。Alkbh1(N6-mA去甲基酶)缺陷的小鼠由于植入后胚胎失去印迹控制和严重的胎盘发育缺陷而在子宫中死亡。有趣的是，ALKBH1缺陷的牛胚胎已经损害了滋养层干细胞的出现和囊胚的形成。此外，从人类多能干细胞分化而来的滋养层细胞中，N6-mA的丰度非常高，为研究N6-mA在人类胎盘中的作用提供了一个很好的模型。这些结果表明，这个新的表观遗传标记在不同真丝虫的滋养层发育过程中具有保守和独特的作用。然而，N6-mA功能的分子机制及其在滋养外胚层和滋养层发育中的作用仍然不清楚。总体假设是，N6-mA代表一种新的表观遗传标记，它调节基因表达网络，指导决定滋养层细胞的命运。这项研究的总体目标是对小鼠和牛胚胎的滋养层细胞以及人类滋养层细胞培养模型进行系统的比较研究，重点研究N6-mA在滋养层细胞形成、基因表达和表观遗传重编程中的作用。提出了三个独立但又与智力相关的具体目标。目的1通过建立滋养层细胞特异性条件性基因敲除小鼠模型，研究N6-mA在滋养层细胞系分化和印记基因调控中的作用。目的2研究N6-mA在牛胚胎模型中滋养层干细胞出现和囊胚形成中的作用。目的3将利用人类干细胞来源的滋养层细胞分化模型来建立N6-mA在人胎盘滋养层细胞形成中的作用。该项目利用江实验室在胚胎/滋养层发育方面的专业知识和肖实验室在表观遗传调控方面的专业知识来解决滋养层谱系发育的基本问题。这项研究具有创新性，因为它a)提供了一个新的DNA甲基化标记在滋养层谱系形成中的作用，b)揭示了印记基因的表观遗传调控，这是胎盘发育中的一个长期问题，以及c)建立了新的、可比较的胚胎和细胞培养模型，以了解真核生物滋养层细胞命运的保守和独特的表观遗传调控。这项拟议的项目意义重大，因为它将提高我们对早期胚胎丢失和与异常胎盘相关的临床生殖障碍的分子基础的理解。

Trophoblasts play an essential role in communication between the fetus and the mother. Failure to accurately establish the trophoblast lineage results in developmental arrest prior to the blastocyst stage, implantation failure, early miscarriage, and placental abnormalities. Recently, we made the first discovery of a novel DNA methylation on N6-adenine (N6-mA) in mammalian genomes (Wu et al., Nature, 2016; Xie et al., Cell, 2018). Our most recent preliminary studies have shown that N6-mA is predominantly present in the trophectoderm/trophoblast lineage of early embryos, and is conserved in the mouse and bovine and probably across all mammals. Alkbh1 (N6-mA demethylase)-deficient in mice die in utero due to loss of imprinted control and severe placental development defects in post-implantation embryos. Interestingly, ALKBH1 deficient bovine embryos have impaired trophoblast stem cell emergence and blastocyst formation. In addition, N6-mA abundance is remarkably high in trophoblast cultures differentiated from human pluripotent stem cells, providing an excellent model for studying N6-mA in human placentation. These results indicated that this novel epigenetic mark has both conserved and unique roles in trophoblast development in different eutherians. However, the molecular mechanism underlying N6-mA function, and its role in trophectoderm and trophoblast development remain elusive. The overall hypothesis is that N6-mA represents a novel epigenetic mark that regulates gene expression network directing determination of trophoblast cell fate. The overall objective of the proposed studies is to conduct a systematic, comparative study on trophoblast from mice and bovine embryos, as well as human trophoblast cell culture models, focusing on the role of N6-mA in trophoblast formation, gene expression and epigenetic reprogramming. Three independent and yet intellectually-related specific aims are proposed. Aim 1 will characterize N6-mA function in trophoblast lineage differentiation and imprinted gene control with a trophoblast-specific conditional knockout mouse model. Aim 2 will investigate the role of N6-mA in trophoblast stem cell emergence and blastocyst formation in a bovine embryo model. Aim 3 will utilize human stem cell-derived trophoblast differentiation models to establish N6-mA functions in human placental trophoblast formation. This project leverages the expertise of the Jiang laboratory in embryo/trophoblast development and the expertise of the Xiao laboratory in epigenetic regulation to address fundamental questions in trophoblast lineage development. This study is innovative because it a) provides insight into the role of a novel DNA methylation mark in trophoblast lineage formation, b) sheds new light on epigenetic regulation of imprinted genes, a longstanding issue in placental development, and c) establishes novel, comparative embryo and cell culture models to understand the conserved and unique epigenetic regulation of trophoblast cell fate in eutherians. The proposed project is significant because it will improve our understanding of the molecular basis of early embryonic loss and clinical reproductive disorders that are associated with abnormal placentation.