Cohesin-mediated loop extrusion and enhancer-associated factors additively contribute to Sox2 looping with its distal enhancer

Sox2 expression is regulated by a distal enhancer cluster known as the Sox2 control region (SCR). Although Sox2 and the SCR exhibit strong interactions in chromosome conformation capture experiments, acute depletion of cohesin only mildly affects Sox2 transcription. Here we show that continuous restarting of loop extrusion, but not stalling of cohesin at CTCF sites, is involved in the maintenance of the Sox2 –SCR loop. While depletion of cohesin or CTCF has only a mild impact on Sox2 gene expression, stabilizing cohesin on the DNA by depleting the cohesin release factor WAPL results in a rapid decrease in expression. Furthermore, using genome editing, we show that a weak enhancer located downstream from the Sox2 gene renders Sox2 expression insensitive to the loss of cohesin stalling at CTCF sites. Finally, through targeted and pleiotropic inhibition of the SCR, we demonstrate that the Sox2 –SCR loop is a multicomponent system that depends on cohesin-mediated loop extrusion and enhancer-associated factors. Together, our findings highlight the intricate interplay between enhancer activity and loop extrusion in the establishment and maintenance of chromatin loops in the Sox2 locus.