An endogenous retinoic acid response element enhancer controls Cyp26a1-mediated retinoic acid clearance during embryonic stem cell differentiation.

Abstract

Precise control of retinoic acid (RA) signaling is essential for embryonic development, as fluctuations in RA availability can perturb cell fate determination and contribute to severe developmental abnormalities. Yet how inducible RA clearance is encoded within endogenous chromatin during pluripotent cell differentiation remains poorly understood. Here, using RA-induced differentiation of mouse embryonic stem cells (mESCs), we identify Cyp26a1 as one of the most strongly induced genes during this fate transition. Among genes involved in RA synthesis and metabolism, Cyp26a1 is distinguished by the formation of a highly RA-responsive distal enhancer. This enhancer contains functional RA response elements (RAREs) and is activated by ligand-dependent RAR/RXR binding, p300 recruitment, and H3K27ac deposition, accompanied by increased enhancer-promoter interaction. Genetic deletion of the enhancer or its core RARE motifs markedly impairs Cyp26a1 induction, demonstrating the requirement of this cis-regulatory module for full RA-responsive transcriptional activation. Single-cell transcriptomic profiling further shows that disruption of the enhancer-RARE module, or loss of Cyp26a1 itself, alters RA-guided differentiation trajectories, reducing neuroectodermal progression while aberrantly expanding undifferentiated and mesenchymal-like populations. Together, our findings define an endogenous enhancer-RARE module that uniquely governs Cyp26a1-mediated RA clearance. By constraining RA availability during differentiation, this cis-regulatory mechanism functions as a crucial epigenetic checkpoint to stabilize developmental signaling and ensure the precise execution of proper cell fate trajectories.