Reduced Oct4 Expression Directs a Robust Pluripotent State with Distinct Signaling Activity and Increased Enhancer Occupancy by Oct4 and Nanog

Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4+/− ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation. •Reduced Oct4 results in homogeneous transcription factor level and delayed differentiation kinetics•Oct4+/− ESCs show increased Oct4 and Nanog binding at pluripotency enhancers•Oct4+/− ESCs exhibit increased Wnt, phospho-STAT3, and LIF sensitivity•Reduced Oct4 levels robustly maintain ESC pluripotency without BMP/serum/2i Reduced Oct4 expression leads to increased Oct4 and Nanog binding to key pluripotency network sites, altered signaling responses that favor self-renewal, and a stabilized pluripotent state that inhibits differentiation.