Three Transcription Factor Functions Empower Formative Transition from Naïve Pluripotency [ChIP-Seq]

The gene regulatory network in naïve mouse embryonic stem cells (ESC) is reconfigured to enable lineage commitment. Tcf3 sanctions rewiring to formative pluripotency by suppressing components of the ESC transcription factor circuitry. However, Tcf3 depletion only delays, and does not prevent transition. Here we delineate major contributions of Ets-family transcription factor Etv5 and the repressor Rbpj. ERK signalling triggers genome relocation of Etv5 to commission formative pluripotency enhancers. Concomitant up-regulation of Rbpj prevents reversion by repressing potent naïve factors, Nanog and Tbx3. Triple deletion of Etv5, Rbpj and Tcf3 disables naïve ESC, such that they remain undifferentiated and locked in self-renewal even in the presence of differentiation stimuli. Thus, pluripotency dynamics are driven by combined action of two repressors that respectively dissolve and extinguish the naive network, and an activator that initiates transcription of formative network genes. Similar tripartite modality might be a general requirement for robust cell state transitions. ChIP-seq of Etv5-3xFlag knock-in (KI) RGd2 ESCs cultured in 2i and withdrawn from 2i inhibitors for 16hrs (N16h) using M2-Flag Antibody (Sigma). 2 Etv5-3xFlag knock-in (KI) clonal ESC lines (clones 20 and 32) were used as biological replicates per condition. Wild type RGd2 ESCs were used as negative control. ChIP-seq of RGd2 ESCs cultured in 2i and withdrawn from 2i inhibitors for 16hrs (N16h), and 25hrs (N25h) FACS-sorted for Rex1-GFPd2 (Rex1-high, Rex1-low) for H3K4me3.