RNA Polymerase II pausing enhances estrogen receptor-alpha binding on promoters in RNA-dependent manner

Regulated pausing of RNA polymerase II (Pol II) is essential for enabling rapid and coordinated transcriptional responses to signalling cues. Pausing also contributes to the formation of nucleosome-free regions with the help of chromatin remodellers. However, if these nucleosome free regions engage with transcription factors to stimulate the transcription potential of paused promoters is not known. In this study, we demonstrate that ligand-induced estrogen receptor-alpha (ERα) binding is stabilized at Pol II-paused sites. This stabilization results from an increased dwell time of ERα on chromatin as revealed by single-particle tracking experiments. Notably, short chromatin-associated RNAs, generated by the stalled Pol II, contribute to enhancing ERα binding at paused promoters. We also observe that pausing increases histone H3K27 acetylation (H3K27ac) levels, which primes paused promoters for robust transcriptional activation upon release. Collectively, these findings suggest that paused Pol II plays a central role in enhancing transcription factor binding through an RNA-dependent mechanism. This, in turn, results in a more vigorous transcriptional response following pausing release, thus contributing to the fine-tuning of ERα-mediated gene regulation.