A specific pluripotency-associated eRNA controls Nanog locus by shaping the epigenetic landscape and stabilizing enhancer–promoter interaction

Despite a plethora of studies exploring the transcriptional regulation of the Nanog gene, the role of the enhancer RNAs (eRNAs) derived from Nanog-interacting super-enhancers (SEs) remains under-investigated. In the present study, we examined the functional role of the eRNAs transcribed from the −5 kb Nanog SE in mouse embryonic stem cells (mESCs) and found that an eRNA, here defined as −5KNAR, was essential to maintain the Nanog locus in an epigenetically active configuration, thereby ensuring pluripotency. We found that the here identified −5KNAR functionally interacts with the RAD21 protein, suggesting a role in stabilizing a cohesin complex at the Nanog locus, ensuring the generation and maintenance of an enhancer–promoter loop. Silencing of −5KNAR caused a cascade of events, including the generation of a DNA methylation wave (likely spreading from a single methylated CpG site), substantial chromatin remodeling, and loss of the enhancer–promoter loop, inducing Nanog silencing and mESC differentiation. Under these conditions, exogenous re-expression of Nanog was unable to restore either the endogenous Nanog expression or the enhancer–promoter interaction, suggesting that, at hierarchical level, the expression of the −5KNAR plays a prominent role in maintaining the pluripotency in mESCs.