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

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.

表观遗传机制调控发育过程中的细胞命运决定，但DNA甲基化如何与染色质结构、三维DNA折叠协同，以执行细胞类型特异性基因表达程序，目前仍不甚明晰。本研究利用5-甲基胞嘧啶（5-methyl cytosine）及其氧化衍生物（5-羟基胞嘧啶、5-甲酰基胞嘧啶与5-羧基胞嘧啶）缺陷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。对DNA甲基转移酶（DNA methyltransferase, Dnmt）或十十一易位酶（Ten-eleven-translocation, Tet）的活性进行遗传敲除后，二者所诱导的失调基因集整体差异显著，揭示了DNA胞嘧啶甲基化循环不同分支受扰动时产生的不同转录缺陷。出乎意料的是，我们发现阻断DNA甲基化或其氧化过程会干扰关键增强子的特征，包括染色质可及性、增强子标志性组蛋白修饰，以及与潜在靶基因之间的远程染色质相互作用。除了影响多能干细胞中特定基因的转录外，我们还观察到，在发育后期必需的关键谱系特化基因相关的调控元件处，增强子预激活功能受损，包括三维相互作用的丧失——这一点我们通过关键造血基因Klf1与Lyl1得到了验证。与之相一致的是，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。本研究揭示了多能性退出阶段DNA甲基化动态更新的全新功能：其可建立并维持染色质状态，通过表观遗传机制预激活增强子，为后续发育细胞分化过程中的增强子激活奠定基础。整体实验设计：将小鼠胚胎干细胞（mESC，培养于2i/Lif培养基中）分化为上胚层样干细胞（EpiLC）或拟胚体（EB）。本研究使用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。