Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes

Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (−45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the −45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the −45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs. [Display omitted] •Genomic editing reveals functional differences in super-enhancer regulation of Nanog•−45 enhancer regulates both nearest neighbor genes, Nanog and Dppa3•−45 eRNAs specifically regulate Dppa3 by stabilizing chromatin looping Super-enhancers are tissue specific cis-regulatory elements that are transcribed and regulate genes critical to cell identity. Blinka et al. show that three neighboring super-enhancers differentially regulate the core pluripotency transcription factor Nanog, and eRNAs produced at one distal enhancer specifically activate a different neighboring gene by stabilizing chromatin looping.