Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and physical proximity

Enhancers are often located tens to hundreds of kilobases away from the genes they regulate. How they exert this regulation in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we have used the activation of estrogen receptor-dependent enhancers in the breast cancer cell line MCF-7 as a model system to study enhancer-promoter communication. This system allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine both enhancer-promoter proximity by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation by Capture-C. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate the roles of enhancer-bound protein complexes and transcription in enhancer-promoter contact frequencies and proximity. Our work further emphasizes that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one, and is modulated by the biochemical and biophysical context of the loci under study. ### Competing Interest Statement The authors have declared no competing interest.