Understanding DNA methylation reprogramming dynamics during preimplantation development using single-cell sequencing

项目总结 在哺乳动物植入前发育期间，最戏剧性的表观遗传学事件之一是全球 从亲本基因组中抹去DNA甲基化(5-甲基胞嘧啶或5mC)。这种重新编程是 对重置配子的甲基化状态和恢复多能细胞的发育潜能至关重要 胚泡。阐明调节5mC重编程的分子机制是获得 更深入地了解正常的胚胎发育，以及更好地理解异常的5mC模式和 与辅助生殖技术(ART)相关的印记障碍。虽然早期的研究表明 父本基因组通过将5mC转化为5-羟甲基胞嘧啶来进行主动的去甲基化 (5hmC)，母体基因组通过缺乏维持DNA而经历被动的去甲基化 甲基转移酶(Dnmt1)在复制过程中的活性，最近的研究表明，5mC擦除 来自亲本基因组的是这两个去甲基化途径的组合。解开这些机制 监管5mC擦除一直是一个挑战，因为我们无法直接区分这些 小路。这一限制可以通过在基因组中对5 mC进行全基因组链特异性测量来克服 单细胞。此外，这种表观遗传的重新编程在时间上与第一个细胞命运规范相一致 在胚胎中向滋养外胚层(TE)和内细胞团(ICM)谱系靠拢。然而，目前仍不清楚 5mC重新编程如何影响这一细胞命运决定。为了解决这些问题，在具体目标1中，我们 建议开发一种新的单细胞测序技术，以同时定量5mC链-特异性 与来自同一细胞的mRNA一起。小鼠胚胎干细胞和小鼠的初步实验 胚胎显示，我们可以从同一个细胞中同时检测到5mC和mRNA。来检验我们的核心假设 调节5mC的擦除的机制是特定于父母和阶段的，在特定的目标2中，我们计划 从2-细胞到-细胞杂交小鼠胚胎中5mC的全基因组链特异性分布 胚胎发育的阶段。此外，通过随机数学建模和击倒实验，我们 将阐明不同的分子因素如何调节主动和被动去甲基化途径 植入前发育。最后，虽然细胞间的可变性在各种因素中已显示出偏向 早期卵裂球对TE或ICM谱系的细胞命运潜力，目前尚不清楚5mC 重新编程会调整或强化这些决定。为了解决这一问题，在具体目标3中，我们计划 量化来自同一细胞的5mC和mRNA，以直接关联全基因组链的异质性- 5mC的特定模式影响细胞对称分裂与不对称分裂之间的决定，以及由此产生的 细胞命运的选择。因此，通过开发新的单细胞方法，我们将获得更深层次的机制 了解植入前发育过程中的整体去甲基化动力学，将使我们能够更好地 研究改变了与ART和其他环境压力相关的表观遗传重编程。

PROJECT SUMMARY During mammalian preimplantation development, one of the most dramatic epigenetic events is the global erasure of DNA methylation (5-methylcytosine or 5mC) from the parental genomes. This reprogramming is critical to reset the methylation status of the gametes and restore developmental potency to pluripotent cells in the blastocyst. Elucidating the molecular mechanisms that regulate 5mC reprogramming is central to gaining deeper insights into normal embryonic development as well as to better understand aberrant 5mC patterns and imprinting disorders associated with assisted reproductive technologies (ART). While early studies showed that the paternal genome undergoes ‘active’ demethylation through conversion of 5mC to 5-hydroxymethylcytosine (5hmC) and the maternal genome undergoes ‘passive’ demethylation through a lack of maintenance DNA methyltransferase (Dnmt1) activity during replication, more recent studies have suggested that 5mC erasure from the parental genomes is a combination of these two demethylation pathways. Unraveling the mechanisms that regulate 5mC erasure has been challenging due to our inability to directly distinguish between these pathways. This limitation can be overcome by making genome-wide strand-specific measurements of 5mC in single cells. In addition, this epigenetic reprogramming coincides temporally with the first cell fate specification in the embryo towards the trophectoderm (TE) and inner cell mass (ICM) lineages. However, it remains unclear how 5mC reprogramming influences this cell fate decision. To address these questions, in Specific Aims 1 we propose to develop a novel single-cell sequencing technology to simultaneously quantify 5mC strand-specifically together with mRNA from the same cell. Preliminary experiments in mouse embryonic stem cells and mouse embryos show that we can detect both 5mC and mRNA from the same cell. To test our central hypothesis that the mechanisms regulating the erasure of 5mC are parent- and stage-specific, in Specific Aims 2 we plan to quantify the genome-wide strand-specific distribution of 5mC in hybrid mouse embryos from the 2- to 64-cell stage of embryogenesis. Further, through stochastic mathematical modeling and knockdown experiments, we will elucidate how different molecular factors regulate active and passive demethylation pathways during preimplantation development. Finally, while cell-to-cell variability in a variety of factors have been shown to bias the cell fate potential of early blastomeres towards the TE or ICM lineages, it remains unclear how 5mC reprogramming tunes or reinforces these decisions. To address this question, in Specific Aims 3 we plan to quantify both 5mC and mRNA from the same cell to directly correlate how heterogeneity in genome-wide strand- specific patterns of 5mC influence the decision between symmetric vs. asymmetric cell divisions and the resulting cell fate choices. Thus, through the development of novel single-cell methods we will gain a deeper mechanistic understanding of global demethylation dynamics during preimplantation development that will enable us to better study altered epigenetic reprogramming associated with ART and other environmental stresses in future.