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

How enhancers regulate their target genes 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 use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.Author summary: Investigating the three-dimensional organization of the genome is important for understanding how enhancers regulate their target genes. Commonly, exploring 3D genome organization uses either an imaging-based method—DNA fluorescence in situ hybridization, or molecular chromosome conformation capture methods. Here we use a cell system of nuclear hormone induced enhancer and gene activation to compare what insight into enhancer–gene promoter interaction these methodologies provide. To our surprise, we found that these two methods can produce paradoxical results, with increased enhancer-promoter contact frequencies assayed by chromosome conformation capture occurring in a situation where imaging shows overall decreased enhancer-promoter proximity. We explore what might contribute to these seemingly discrepant data, demonstrating that whereas increased enhancer-promoter contact frequencies require binding of an active transcription factor to the enhancer but are independent of transcription per se, altered enhancer-promoter proximity depends on transcription. Our analyses show the importance of using orthogonal methods to fully explore 3D genome organization.