Dynamic DNA methylation turnover at the exit of pluripotency epigenetically primes gene regulatory elements for hematopoietic lineage specification [PCHIC]. Dynamic DNA methylation turnover at the exit of pluripotency epigenetically primes gene regulatory elements for hematopoietic lineage specification [PCHIC]

Epigenetic mechanisms govern developmental cell fate decisions, but how DNA methylation coordinates with chromatin structure and three-dimensional DNA folding to enact cell-type specific gene expression programmes remains poorly understood. Here, we use mouse embryonic stem and epiblast-like cells deficient for 5-methyl cytosine or its oxidative derivatives (5-hydroxy-, 5-formyl- and 5-carboxy-cytosine) to dissect the gene regulatory mechanisms that control cell lineage specification at the exit of pluripotency. Genetic ablation of either DNA methyltransferase (Dnmt) or Ten-eleven-translocation (Tet) activity yielded largely distinct sets of dysregulated genes, revealing divergent transcriptional defects upon perturbation of individual branches of the DNA cytosine methylation cycle. Unexpectedly, we found that disrupting DNA methylation or oxidation interferes with key enhancer features, including chromatin accessibility, enhancer-characteristic histone modifications, and long-range chromatin interactions with putative target genes. In addition to affecting transcription of select genes in pluripotent stem cells, we observe impaired enhancer priming, including a loss of three-dimensional interactions, at regulatory elements associated with key lineage-specifying genes that are required later in development, as we demonstrate for the key hematopoietic genes Klf1 and Lyl1. Consistently, we observe impaired transcriptional activation of blood genes during embryoid body differentiation of knockout cells. Our findings identify a novel role for the dynamic turnover of DNA methylation at the exit of pluripotency to establish and maintain chromatin states that epigenetically prime enhancers for later activation during developmental cell diversification. Overall design: Mouse embryonic stem cells (mESC, cultured in 2i/Lif) were differentiated to epiblast like stem cells (EpiLC) or embryoid bodies (EB). Four cell lines were used: A triple knock-out of Tet1, Tet2 and Tet3 (TET_KO) and the corresponding parental wild-type line (TET_WT); a triple knock-out of Dnmt1, Dnmt3a and Dnmt3b (DNMT_KO) and the corresponding parental wild-type line (DNMT_WT). Conditions were generally assessed in triplicate.

表观遗传机制（epigenetic mechanisms）调控发育进程中的细胞命运决定，但DNA甲基化如何与染色质结构及三维DNA折叠协同作用，以执行细胞类型特异性基因表达程序，目前仍缺乏深入认知。本研究利用缺失5-甲基胞嘧啶（5-methyl cytosine）或其氧化衍生物（5-羟甲基胞嘧啶、5-甲酰基胞嘧啶及5-羧基胞嘧啶）的小鼠胚胎干细胞及上胚层样细胞，解析多能性退出阶段调控细胞谱系特化的基因调控机制。遗传敲除DNA甲基转移酶（DNA methyltransferase, Dnmt）或Tet蛋白（Ten-eleven-translocation, Tet）的活性后，所产生的失调基因集大体互不重叠，这表明扰动DNA胞嘧啶甲基化循环的单个分支会引发迥异的转录缺陷。出乎意料的是，我们发现破坏DNA甲基化或其氧化过程会干扰关键增强子的核心特征，包括染色质开放性、增强子标志性组蛋白修饰，以及与潜在靶基因间的长程染色质相互作用。除影响多能干细胞中特定基因的转录外，我们还观察到增强子预激活（enhancer priming）受损，包括三维相互作用的丢失，此类变化发生在与发育后期所需关键谱系特化基因相关的调控元件上，我们以关键造血基因Klf1和Lyl1为例进行了验证。与此一致，我们在敲除细胞的拟胚体（embryoid body, EB）分化过程中，观察到血液相关基因的转录激活受损。本研究发现，在多能性退出阶段，DNA甲基化的动态更新在建立并维持染色质状态中发挥了全新作用，此类染色质状态可通过表观遗传机制预激活增强子，以支持发育后期细胞分化过程中的后续基因激活。实验整体设计：将在2i/Lif培养基中培养的小鼠胚胎干细胞（mouse embryonic stem cells, mESC）诱导分化为上胚层样干细胞（epiblast like stem cells, EpiLC）或拟胚体（EB）。本研究共使用4种细胞系：Tet1、Tet2与Tet3三基因敲除细胞系（TET_KO）及其对应的野生型亲本细胞系（TET_WT）；Dnmt1、Dnmt3a与Dnmt3b三基因敲除细胞系（DNMT_KO）及其对应的野生型亲本细胞系（DNMT_WT）。所有实验条件均设置3次生物学重复。