Embryonic stem cell-specific intragenic enhancer RNA essential for NSUN2-mediated stem cell fate regulation

NSUN2 (NOP2/Sun RNA methyltransferase 2), an RNA m5C-methyltransferase responsible for the 5-methylcytosine (m5C) modification in various RNAs, is highly expressed in embryonic stem cells (ESCs) and is essential for multiple biological processes, including regulating the balance between pluripotency and differentiation, as well as brain development. However, the regulatory mechanisms governing Nsun2 expression in ESCs remain largely unknown. Through a comprehensive multi-omics analysis integrating ChIP-seq, GRO-seq, and ATAC-seq, we identified a putative ESC-specific intragenic enhancer of Nsun2, suggesting its role in enhancer-mediated gene regulation in mESCs. The enhancer RNA (eRNA) transcribed from this region was found to significantly regulate Nsun2 expression exclusively in mouse ESCs (mESCs), coinciding with changes in mRNA stability of pluripotency-related genes, suggesting a potential contribution to maintaining pluripotency. Knockdown of this eRNA was associated with reduced Nsun2 expression, diminished m5C methylation activity, and lower expression of pluripotency markers. Furthermore, differentiation assays revealed altered lineage-specific gene expression, supporting the enhancer's role in pluripotency and early differentiation. These findings suggest a potential ESC-specific regulatory mechanism in which an intragenic eRNA may influence Nsun2 expression and function, emphasizing the importance of eRNAs in the epigenetic control of stem cell fate regulation.