Enhancer RNAs stimulate Pol II pause release by harnessing multivalent interactions to NELF

Abstract

Enhancer RNAs (eRNAs) are long non-coding RNAs that originate from enhancers. Although eRNA transcription is a canonical feature of activated enhancers, the molecular features required for eRNA function and the mechanism of how eRNAs impinge on target gene transcription have not been established. Thus, using eRNA-dependent RNA polymerase II (Pol II) pause release as a model, we here investigate the requirement of sequence, structure and length of eRNAs for their ability to stimulate Pol II pause release by detaching NELF from paused Pol II. We find eRNAs not to exert their function through common structural or sequence motifs. Instead, eRNAs that exhibit a length >200 nucleotides and that contain unpaired guanosines make multiple, allosteric contacts with NELF subunits -A and -E to trigger efficient NELF release. By revealing the molecular determinants of eRNA function, our study establishes eRNAs as an important player in Pol II pause release, and it provides new insight into the regulation of metazoan transcription.

The pleiotropic roles of SPT5 in transcription

“…Numerous studies have reported the regulatory role of eRNAs in activating target genes [ 145–147 ]. For instance, eRNAs could interact with NELF to facilitate its eviction, and thus promote the release of paused Pol II from promotor-proximal regions [ 148 , 149 ], in accordance with the increased paused Pol II release upon enhancer activation [ 56 ]. This suggests that there are at least two layers of gene transcriptional control by SPT5: one is through its direct association with Pol II at promoters and gene bodies, and the other is through indirectly influencing target genes by modulating activation of their corresponding enhancers.…”

Enhancer RNAs step forward: new insights into enhancer function

“…eRNAs interact with P-TEFb by directly binding to cyclin T1; the resultant activation of P-TEFb promotes the phosphorylation of NELF, DRB sensitivity inducing factor (DSIF) and Serine 2 of the RNAPII C-terminal domain (CTD), via the pause-releasing kinase activity of CDK9 ( Fujinaga et al, 2004 ; Shii et al, 2017 ; Wada et al, 1998 ; Yamaguchi et al, 1999 ; Zhao et al, 2016 ). P-TEFb-catalysed phosphorylation sites on the NELF-A subunit of the NELF complex overlap with eRNA binding sites identified on this subunit ( Gorbovytska et al, 2022 ). Phosphorylation of such sites is essential for RNAPII pause release ( Lu et al, 2016 ) and is hypothesised to induce a conformational change in NELF conducive to NELF release from the paused elongation complex.…”

“…In line with this, eRNA binding within the vicinity of P-TEFb target sites may trigger a similar conformational change that bypasses P-TEFb activity altogether. Consequently, NELF phosphorylation by P-TEFb and eRNA binding may similarly bestow a negative charge to NELF that facilitates a conformational change and, ultimately, results in dissociation of NELF from the paused elongation complex ( Gorbovytska et al, 2022 ).…”

Enhancer-Mediated Formation of Nuclear Transcription Initiation Domains

“…Phosphorylation of these proteins by pTEFb dissociates NELF and converts DSIF into a positive transcription elongation factor. It was shown that eRNA of a length greater than 200 nucleotides and containing unpaired guanosines interacts with NELF subunits allosterically and causes release from Pol II, thereby stimulating transcription elongation [ 68 ]. Thus, enhancers may contribute to efficient transcription elongation by two distinct mechanisms: the p300/CBP mediated acetylation of H3K27, which recruits Brd4 and, consequently, pTEFb, and eRNA-mediated dissociation of NELF.…”