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.