G-quadruplexes are promoter elements controlling nucleosome exclusion and RNA polymerase II pausing.

Despite their central role in transcription, it has been difficult to define universal sequences associated with eukaryotic promoters. Within the chromatin context, recruitment of transcriptional machinery requires promoter opening, but how DNA elements contribute to this process is unclear. Here we show that G-quadruplex (G4) secondary DNA structures are highly enriched at mammalian promoters. G4s are located at the deepest point of nucleosome exclusion at promoters and correlate with maximum promoter activity. We found that experimental G4s exclude nucleosomes in vivo and in vitro while favouring strong positioning. At model promoters, impairing G4s affected both transcriptional activity and chromatin opening. G4 destabilization also resulted in an inactive promoter state and affected the transition to effective RNA production. Finally, G4 stabilization resulted in global reduction of proximal promoter pausing. Altogether, our data introduce G4s as bona fide promoter elements allowing nucleosome exclusion and facilitating pause-release by RNA polymerase II.