Two unrelated distal genes activated by a shared enhancer benefit from localizing inside the same small topological domain (HiC).

Enhancers are tissue-specific regulatory DNA elements that can activate transcription of genes over distance. Their target genes most often locate in the same topologically associating domain (TAD). TADs are structural entities within chromosomes in which cohesin DNA loop extrusion supports intra-domain DNA contacts and CTCF-bound boundaries serve to halt and concentrate paralogous genes. Enhancers shared by multiple unrelated genes may be more common than those acting on paralogous gene clusters, but are underexplored. Here, we analyzed the interplay between an enhancer and two distal, functionally unrelated, genes residing at opposite domain boundaries. The enhancer stimulated boundary and gene-gene contacts and required cohesin as well as two intact domain boundaries to support gene activation. The two genes preferentially transcribed when spatially together, but did not rely on each other's transcription, nor showed gene competition. Placing them and the enhancer in smaller domains caused their upregulation. Domain boundaries, therefore, can support long-range enhancer-promoter communication inside domains. Boundary proximity thereby matters as the distal enhancer functions more effectively inside smaller domains. We propose that smaller domains have more concentrated cohesin loop extrusion activity to facilitate enhancer action over distance. Overall design: Capture Hi-C data with probes capturing the MYEOV-CCND1 locus of WT, enhancer deleted, MYEOV deleted, CCND1 deleted and CTCF2 deleted K562 cells.