The self‐renewal and differentiation potential of embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. Expression levels of the core pluripotency factor Nanog are tightly regulated by a negative feedback autorepression loop. However, it remains unclear how ESCs perceive NANOG levels and execute autorepression. Here, we show that a dose‐dependent induction of Fgfbp1 and Fgfr2 by NANOG activates autocrine‐mediated ERK signaling in Nanog‐high cells to trigger autorepression. pERK recruits NONO to the Nanog locus to repress transcription by preventing POL2 loading. This Nanog autorepression process establishes a self‐perpetuating reciprocal NANOG‐pERK regulatory circuit. We further demonstrate that this reciprocal regulatory circuit induces pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells. Collectively our data suggest that NANOG induces Fgfr2 and Fgfbp1 to activate ERK signaling in Nanog‐high cells to establish a NANOG‐pERK reciprocal regulatory circuit. This circuit regulates ERK signaling dynamics and Nanog autoregulation in pluripotent cells.

FGF/ERK autocrine signaling induces Nanog heterogeneity, allelic switching, and differentiation in pluripotent cells, which are abolished in the naive-state (2i) by inhibition of MEK1/2 and GSK3?. Surprisingly Nanog autorepression is functional in 2i. The mechanisms activating FGF signaling and its downstream effectors leading to heterogeneity and allelic switching are unknown. We utilized genome-edited fluorescent reporter cell lines of both Nanog promotor and protein to elucidate, a NANOG induced FGF autocrine signaling mediated autoregulatory mechanism. We show that Nanog autoregulation is a cell non-autonomous process initiated by NANOG, but executed by FGF/ERK. FGFR2 differentially activates autoregulation in Nanog-high cells by activating ERK1/2. pERK1/2 recruits NONO (P54nrb) to repress Nanog transcription. Collectively, we show that Nanog-induced FGF autocrine signaling mediated autoregulation is the basis of Nanog heterogeneity and allelic switching. Our data introduces an unexpected reciprocal regulatory mechanism between NANOG and FGF/ERK signaling feeding into each other's heterogeneity.