The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II at pro-differentiation genes [ChIP-Seq]

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid transcription. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ~1,700 TFs with CRISPR loss-of-function perturbations, we found that ZBTB11 and ZFP131 are required to maintain pluripotency in mouse embryonic stem cells (ESCs). ZBTB11 and ZFP131 bind to pro-differentiation genes along with RNA Polymerase II and pausing factor NELF, but without Polycomb repression. Loss of ZBTB11 or ZFP131 leads to a decrease in NELF association, an increase in H3K4me3, transcriptional upregulation of genes associated with three germ layers, and concomitant ESC differentiation. Together, our results suggest ZBTB11 and ZFP131 maintain pluripotency by pausing pro-differentiation genes transcription and present a generalizable framework to maintain cellular potency. Overall design: Bulk RNA-seq was used to characterize gene expression in Wildtype, Zbtb10KO, Zbtb11KO, and Zfp131KO cells grown in the presence of +2i+LIF or -2i+LIF.

在多能干细胞（Pluripotent Cells）中，精妙的激活-抑制平衡维持促分化基因处于可快速转录的就绪状态。特异性抑制促分化基因的转录因子（Transcription Factors, TFs）的具体身份仍未明确。本研究通过CRISPR功能丧失扰动靶向约1700个转录因子（TFs），发现ZBTB11与ZFP131是小鼠胚胎干细胞（Embryonic Stem Cells, ESCs）维持多能性所必需的。ZBTB11与ZFP131可与RNA聚合酶II（RNA Polymerase II）、转录暂停因子NELF结合至促分化基因，却不介导多梳蛋白（Polycomb）的基因抑制作用。敲除ZBTB11或ZFP131会导致NELF结合能力下降、组蛋白H3赖氨酸4三甲基化（H3K4me3）水平升高、三胚层相关基因的转录上调，同时伴随胚胎干细胞（ESCs）分化。综上，本研究结果表明ZBTB11与ZFP131可通过暂停促分化基因的转录维持细胞多能性，并提出了一个可推广的维持细胞潜能的研究框架。实验整体设计：本研究采用批量RNA测序（Bulk RNA-seq），对在+2i+LIF或-2i+LIF培养条件下生长的野生型（Wildtype）、Zbtb10敲除（Zbtb10KO）、Zbtb11敲除（Zbtb11KO）及Zfp131敲除（Zfp131KO）细胞的基因表达特征进行了表征。