Interplay between CTCF boundaries and a super enhancer controls cohesin extrusion trajectories and gene expression

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation. [Display omitted] •An enhancer initiates loop extrusion trajectories that delineate activatable genes•Enhancer insulation does not depend strictly on looping between its CTCF boundaries•A loop-engaged inverted CTCF boundary can also block cohesin extrusion trajectories•A strong CTCF boundary permits rare but productive enhancer-promoter contacts The three-dimensional structure of the genome, organized by architectural proteins CTCF and cohesin, has a strong influence on how genes are expressed. Here, Vos et al. allele-specifically dissect an active genomic region to reveal how an enhancer and CTCF boundaries organize cohesin extrusion trajectories, build structural domains, and regulate genes.