RRBS profiling and Transcriptome profiling of Stat3 wild-type, Stat3 knock-out, Dnmt3a/b wild-type and Dnmt3a/b knock-out mouse embryonic stem cells (mES cells)

Naive pluripotent epiblast cells of the preimplantation murine embryo and their in vitro counterpart, embryonic stem (ES) cells, have the capacity to give rise to all cells of the adult. Such developmental plasticity is associated with global genome hypomethylation. It is unclear whether genome methylation is dynamically regulated only via differential expression of DNA methyltransferases (DNMTs) and Ten-eleven Translocation (TET) enzymes, which oxidase methylated DNA. Here we show that LIF/Stat3 signalling induces genomic hypomethylation via metabolic reconfiguration. In Stat3-/- ES cells we observed decreased alpha-ketoglutarate (ɑKG) production from reductive Glutamine metabolism, leading to decreased TET activity, increased Dnmt3a/b expression and to a global increase in DNA methylation. Notably, genome methylation is dynamically controlled by simply modulating αKG availability, mitochondrial activity or Stat3 activation in mitochondria, indicating an effective crosstalk between metabolism and the epigenome. Stat3-/- ES cells also show increased methylation at Imprinting Control Regions accompanied with differential expression of >50% of imprinted genes. Single-cell transcriptome analysis of Stat3-/- embryos confirmed dysregulated expression of Dnmt3a/b, Tet2, and imprinted genes in vivo. Our results reveal that the LIF/Stat3 signal bridges the metabolic and epigenetic profiles of naive pluripotent cells, ultimately controlling genome methylation and imprinted gene expression. Several imprinted genes regulate cell proliferation and are often misregulated in tumours. Moreover, a wide range of cancers display Stat3-overactivation, raising the possibility that the molecular module we described here is exploited under pathological conditions.

着床前小鼠胚胎的初始态多能上胚层细胞，及其体外培养的对应物——胚胎干细胞（embryonic stem, ES细胞），具备分化为成体所有细胞类型的潜能。这种发育可塑性与全基因组低甲基化状态紧密关联。目前学界尚未明确，基因组甲基化的动态调控是否仅依赖于DNA甲基转移酶（DNA methyltransferases, DNMTs）与氧化甲基化DNA的Ten-eleven易位（Ten-eleven Translocation, TET）酶的差异表达。本研究证实，LIF/Stat3信号通路可通过代谢重编程诱导基因组低甲基化。在Stat3纯合敲除（Stat3-/-）的ES细胞中，我们观察到还原性谷氨酰胺代谢途径生成的α-酮戊二酸（α-ketoglutarate, αKG）水平下降，继而导致TET酶活性降低、Dnmt3a/b基因表达上调，并引发全基因组DNA甲基化水平整体升高。值得关注的是，仅通过调控αKG的可用性、线粒体活性，或线粒体中的Stat3激活状态，即可动态改变基因组甲基化水平，这表明代谢过程与表观基因组之间存在高效的串扰。Stat3-/- ES细胞的印记控制区域（Imprinting Control Regions）甲基化水平也显著升高，并伴随超过50%的印记基因出现差异表达。对Stat3-/-小鼠胚胎的单细胞转录组分析（single-cell transcriptome analysis）进一步证实，体内Dnmt3a/b、Tet2以及印记基因的表达均发生失调。本研究结果揭示，LIF/Stat3信号通路能够连接初始态多能细胞的代谢特征与表观遗传谱，最终实现对基因组甲基化水平及印记基因表达的调控。部分印记基因可调控细胞增殖，且常在肿瘤组织中出现表达异常。此外，多种癌症均表现出Stat3过度激活的表型，这提示我们所阐释的分子调控模块，可能在病理状态下被异常激活利用。