Two super-enhancers regulate Nanog expression through distinct mechanisms [45del]

Enhancers are critical regulators of gene expression which are historically defined using plasmid-based approaches. Recent work has emphasized that enhancers operate by alleviating promoter-proximal pause release, implying that a single enhancer operates on a gene at any given moment. By contrast, our previous work postulated that two super-enhancers (SEs) may simultaneously operate on a single gene. To address this discrepancy we employed genomic editing, single cell analyses, and genome-wide approaches to assess how two SEs regulate Nanog expression in embryonic stem cells. We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We also demonstrate that two SEs simultaneously operate on Nanog by regulating different phases of transcription, with one SE operating by recruiting and/or initiating RNA Polymerase II, and the second promoting transcriptional elongation after promoter-proximal pause release. These findings imply SEs can use different mechanisms to regulate expression. Overall design: RNA-seq of 2 conditions, 3 replicates of each