The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II at pro-differentiation genes [scRNA-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 binding, 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. Single-cell RNA-seq was used to characterize gene expression in mixed populations of mES cells containing H2B::GFP expressing wildtype cells and Zbtb11 or Zfp131 knockout cells.