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

Despite a plethora of studies explored the transcriptional regulation of Nanog gene, the role of the eRNAs derived from Nanog interacting super-enhancers remains under-investigated. In the present study, we examined the functional role of the eRNAs transcribed from the -5kb Nanog super-enhancer in mouse embryonic stem cells (mESCs) and found that an antisense eRNA was essential to maintain the Nanog locus in an epigenetic active configuration, thus ensuring pluripotency. We found that the here identified antisense eRNA functionally interact with Rad21 protein, stabilizing the cohesin complex at the Nanog locus, ensuring the generation and maintenance of an enhancer-promoter loop. Silencing of -5kb antisense eRNA caused a cascade of events including the generation of a DNA methylation wave, substantial chromatin remodeling and loss of promoter-enhancer loop, inducing Nanog silencing and mESCs differentiation. In these conditions, exogenous Nanog re-expression was not able to restore neither the endogenous Nanog expression nor the enhancer-promoter interaction suggesting that, at hierarchical level, the expression of the antisense eRNA plays a prominent role in maintaining the pluripotency in mouse embryonic stem cells.