Enhancer RNAs harness multivalent interactions to detach NELF from paused Pol II [GROSEQ, eCLIPSEQ, CHIPSEQ]

Enhancer RNAs (eRNAs) are a class of non-coding RNAs that originate from enhancers. Although eRNA transcription is a canonical feature of functionally activated enhancers in various organisms and cell types, its functionality in gene regulation has not been firmly established. This is in part due to a lack of knowledge regarding the molecular features required for eRNA function. Using the eRNA-dependent regulation of RNA polymerase II (Pol II) pause release as a model, we examined the requirement of sequence, structure and length for the ability of eRNAs to detach NELF from paused Pol II. We found no common structural or sequence motif that dictates eRNA function. Instead, efficient NELF release requires a single eRNA molecule to make multiple contacts with several NELF subunits as well as the presence of unpaired guanosine nucleosides. By revealing the molecular determinants for eRNA function, our study mechanistically links eRNAs to Pol II pause release and provides new insight into the regulation of metazoan transcription. Overall design: GRO-, eCLIP-, or ChIP-seq profiles were generated from primary mouse cortical neurons at DIV7, comparing untreated neurons to neurons for different length of time.