DNA Methylation at N6-Adenine in Placental Trophoblast Development

胎盘滋养层发育中 N6-腺嘌呤 DNA 甲基化

• 批准号: 10705982
• 负责人: Zongliang Jiang
• 金额: $ 46.91万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705982
• 关键词: DNA; Methylation; N6; Adenine; Placental

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.

滋养层细胞在胎儿和母亲之间的交流中起着重要作用。未能准确建立滋养层谱系导致胚泡期前发育停滞、着床失败、早期流产和胎盘异常。最近，我们首次发现哺乳动物基因组中N6-腺嘌呤（N6-mA）上的新DNA甲基化（Wu et al.，Nature，2016; Xie等人，Cell，2018）。我们最近的初步研究表明，N6-mA主要存在于早期胚胎的滋养外胚层/滋养层谱系中，并且在小鼠和牛以及可能在所有哺乳动物中是保守的。Alkbh 1（N6-mA脱甲基酶）缺陷的小鼠在子宫内死亡，原因是印记控制的丧失和植入后胚胎中严重的胎盘发育缺陷。有趣的是，ALKBH 1缺陷的牛胚胎具有受损的滋养层干细胞出现和囊胚形成。此外，N6-mA丰度是显着高的滋养层培养分化的人多能干细胞，提供了一个很好的模型，研究N6-mA在人类胎盘。这些结果表明，这种新的表观遗传标记在不同的真兽类滋养层发育中既保守又独特。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难以捉摸。总的假设是，N6-mA代表一种新的表观遗传标记，调节基因表达网络指导决定滋养层细胞的命运。拟议研究的总体目标是对小鼠和牛胚胎的滋养层以及人类滋养层细胞培养模型进行系统的比较研究，重点关注N6-mA在滋养层形成，基因表达和表观遗传重编程中的作用。提出了三个独立但与智力有关的具体目标。目的1将通过滋养层特异性条件性基因敲除小鼠模型来表征N6-mA在滋养层细胞谱系分化和印迹基因控制中的功能。目的2研究N6-mA在牛胚胎滋养层干细胞出现和囊胚形成中的作用。目的3利用人干细胞源性滋养层细胞分化模型研究N6-mA在人胎盘滋养层细胞形成中的作用。该项目利用了Jiang实验室在胚胎/滋养层发育方面的专业知识和Xiao实验室在表观遗传调控方面的专业知识，以解决滋养层谱系发育中的基本问题。这项研究是创新的，因为它a）提供了一种新的DNA甲基化标记在滋养层谱系形成中的作用的见解，B）揭示了印记基因的表观遗传调控的新观点，这是胎盘发育中的一个长期问题，以及c）建立了新的比较胚胎和细胞培养模型，以了解真兽目动物滋养层细胞命运的保守和独特的表观遗传调控。该项目具有重要意义，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疾病的分子基础的理解。