A bipartite function of ESRRB can integrate signaling over time to balance self-renewal and differentiation.

Cooperative DNA binding of transcription factors (TFs) integrates the cellular context to support cell specification during development. Naive mouse embryonic stem cells are derived from early development and can sustain their pluripotent identity indefinitely. Here, we ask whether TFs associated with pluripotency evolved to directly support this state or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is absent, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB allows quantitative titration of this differentiation, and in silico modeling of bipartite ESRRB activity suggests it safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow ex vivo expansion of immortal stem cells. A record of this paper's transparent peer review process is included in the supplemental information.