Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

Abstract Self‐renewal of embryonic stem cells (ESCs) cultured in LIF/fetal calf serum (FCS) is incomplete with some cells initiating differentiation. While this is reflected in heterogeneous expression of naive pluripotency transcription factors (TFs), the link between TF heterogeneity and differentiation is not fully understood. Here, we purify ESCs with distinct TF expression levels from LIF/FCS cultures to uncover early events during commitment from naive pluripotency. ESCs carrying fluorescent Nanog and Esrrb reporters show Esrrb downregulation only in Nanog low cells. Independent Esrrb reporter lines demonstrate that Esrrb negative ESCs cannot effectively self‐renew. Upon Esrrb loss, pre‐implantation pluripotency gene expression collapses. ChIP‐Seq identifies different regulatory element classes that bind both OCT4 and NANOG in Esrrb positive cells. Class I elements lose NANOG and OCT4 binding in Esrrb negative ESCs and associate with genes expressed preferentially in naive ESCs. In contrast, Class II elements retain OCT4 but not NANOG binding in ESRRB‐negative cells and associate with more broadly expressed genes. Therefore, mechanistic differences in TF function act cumulatively to restrict potency during exit from naive pluripotency.