Cuomo Mariella, Costabile Davide, Della Monica Rosa, Buonaiuto Michela, Trio Federica, De Riso Giulia, Visconti Roberta, Chiariotti Lorenzo
Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II", Via S. Pansini 5, Naples 80131, Italy.
CEINGE Advanced Biotechnologies "Franco Salvatore", Naples 80145, Italy.
Nucleic Acids Res. 2025 Apr 10;53(7). doi: 10.1093/nar/gkaf274.
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.
尽管有大量研究探索Nanog基因的转录调控，但源自与Nanog相互作用的超级增强子（SEs）的增强子RNA（eRNAs）的作用仍未得到充分研究。在本研究中，我们检测了从小鼠胚胎干细胞（mESCs）中-5 kb Nanog SE转录的eRNAs的功能作用，发现一种eRNA（此处定义为-5KNAR）对于将Nanog基因座维持在表观遗传活性状态至关重要，从而确保多能性。我们发现此处鉴定的-5KNAR在功能上与RAD21蛋白相互作用，表明其在稳定Nanog基因座处的黏连蛋白复合物中发挥作用，确保增强子-启动子环的形成和维持。-5KNAR的沉默引发了一系列事件，包括DNA甲基化波的产生（可能从单个甲基化的CpG位点扩散）、大量染色质重塑以及增强子-启动子环的丢失，导致Nanog沉默和mESC分化。在这些条件下，外源性重新表达Nanog无法恢复内源性Nanog表达或增强子-启动子相互作用，这表明在层级水平上，-5KNAR的表达在维持mESCs的多能性中起着重要作用。
