Three Transcription Factor Functions Empower Progression from Naïve to Formative Pluripotency [RNA-Seq]

The gene regulatory network in naïve mouse embryonic stem cells (ESCs) must be reconfigured for lineage competence. Tcf3 enables rewiring to formative pluripotency by repressing components of the ESC transcription factor circuitry. However, elimination of Tcf3 only delays, and does not prevent, state transition. Here we delineate distinct contributions of the Ets-family transcription factor Etv5 and the repressor Rbpj. Downstream of Erk1/2 signalling, Etv5 activates enhancers for formative pluripotency. Concomitant up-regulation of Rbpj ensures irreversible exit from the naïve state by extinguishing reversal factors, Nanog and Tbx3. Triple deletion of Etv5, Rbpj and Tcf3 incapacitates ESCs, such that they remain undifferentiated and locked in self-renewal even in the presence of differentiation stimuli. Thus, pluripotency progression is driven hierarchically by two repressors, that respectively dissolve and extinguish the naive network, and an initiator that commissions the formative network. Similar tripartite action may be a general mechanism for efficient cell transitions. Overall design: RNA-seq analysis of parental Rex1-GFPd2 ES cells (RGd2), and deletion mutants generated in this background (Etv5-KO, RbpJ-KO, Etv5-RpbJ-dKO, Etv5-RbpJ-Tcf3-tKO) cultured in 2i, N2B27 or supplemented with Chiron, 3 biological replicates per condition.

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Kalkan T, Bornelöv S, Mulas C, Diamanti E, Lohoff T, Ralser M, Middelkamp S, Lombard P, Nichols J, Smith A