DNA affinity driven chromatin dynamics determine Estrogen Receptor α transcriptional activity independent of RNA binding

RNA binding is emerging as a shared feature of many transcription factors, potentially influencing gene regulation. Here, we dissect the respective contributions of DNA and RNA binding on the behavior of estrogen receptor alpha (ERα). Using a panel of separation-of-function mutants with graded defects in RNA or DNA binding affinity, we show that DNA interactions govern ERα chromatin localization and transient residence times, whereas disruption of RNA binding does not alter chromatin residency dynamics. Chromatin association correlates with gene activation, as mutations that impair RNA-binding affinity had modest, locus-specific effects on ERα-mediated transcription, while reductions in DNA-binding affinity were sufficient to broadly abolish transcriptional outcomes. Together, this work demonstrates that RNA binding has targeted effects on ERα’s activity as a transcription factor, while DNA binding is the primary driver of hormone-dependent transcriptional regulation, correlated with the increase in the transient residence binding time of chromatin-associated ERα.