A specific pluripotency-associated eRNA controls Nanog locus by shaping the epigenetic landscape and stabilizing enhancer–promoter interaction
Mariella Cuomo, Davide Costabile, Rosa Della Monica, Michela Buonaiuto, Federica Trio, Giulia De Riso, Roberta Visconti, Lorenzo Chiariotti
Abstract
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.