(ATAC-seq_and_ChIP-seq) Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency. (ATAC-seq_and_ChIP-seq) Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency

Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here we identify Spic, a member of the ETS family of transcription factors, as a specific marker of ground state pluripotency. We show that Spic is rapidly induced in ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL), and in response to MEK/ERK inhibition. ChIP-seq analysis demonstrated that Spic binds to enhancer elements that are associated with pluripotency genes. Interaction proteomics and genomic profiling confirmed that SPIC interacts with NANOG and stabilizes its binding to chromatin in 2iL-ESCs. Additional gain of function and loss of function experiments revealed that Spic controls genes involved in one carbon (1C) metabolism, Bhmt, Bhmt2, and Dmgdh, and the flux of SAM-to-SAH in 2iL-ESCs. By maintaining low levels of SAM, Spic controls the level of H3K4me3 and H3R17me2 histone methylation in ground state ESCs. Our data highlight the role of uncharacterized axillary transcription factors that link cellular metabolism to epigenetic regulation in ground state pluripotency. Overall design: ATAC-seq and ChIP-seq of SPIC and NANOG in Spic-KO, -OE, and -WT mouse ESCs

解析胚胎干细胞（embryonic stem cells, ESCs）的表观遗传调控机制，对于干细胞生物学与发育生物学具有核心重要性。本研究鉴定出ETS家族转录因子成员Spic，可作为基态多能性的特异性标志物。研究发现，在添加糖原合成激酶3（glycogen synthase kinase 3, GSK3）抑制剂、丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase, MEK）抑制剂与白血病抑制因子（leukemia inhibitory factor, LIF）的培养体系（2iL）中，以及经MEK/ERK抑制处理后，ESCs内的Spic会被快速诱导表达。染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）分析显示，Spic可结合至与多能性基因相关的增强子区域。相互作用蛋白质组学与基因组谱分析证实，在2iL培养的ESCs中，SPIC可与NANOG发生相互作用，并稳定后者与染色质的结合。额外的功能获得与功能缺失实验表明，在2iL-ESCs中，Spic可调控参与一碳（1C）代谢的基因（Bhmt、Bhmt2与Dmgdh），以及S-腺苷甲硫氨酸（S-adenosylmethionine, SAM）向S-腺苷同型半胱氨酸（S-adenosylhomocysteine, SAH）的代谢流。通过维持较低的SAM水平，Spic可调控基态ESCs中的H3K4me3与H3R17me2组蛋白甲基化水平。本研究数据凸显了一类尚未被表征的辅助转录因子的功能——这类因子可在基态多能性状态下，将细胞代谢与表观遗传调控关联起来。实验整体设计：对Spic基因敲除（knockout, KO）、过表达（overexpression, OE）与野生型（wild type, WT）小鼠ESCs中的SPIC与NANOG开展测序法转座酶可及性染色质分析（assay for transposase-accessible chromatin using sequencing, ATAC-seq）与染色质免疫共沉淀测序（ChIP-seq）分析。