Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements regulate gene transcription and chromatin topology is not clearly defined, yet these processes are intimately linked to cell lineage specification during development. Through allele-specific genome editing and chromatin interaction analysis of the Sox2 locus in mouse embryonic stem cells, we found a striking decoupling of transcriptional control and chromatin architecture. We trace nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletion has no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream of the Sox2 enhancer, is conversely widely distributed over multiple transcription factor bound regions and maintained in a CTCF independent manner. Furthermore, disruption of promoter-enhancer interactions via induced ectopic chromatin loop formation has no effect on Sox2 expression. These findings indicate many transcription factors are involved in modulating chromatin architecture independently from CTCF. Allele-specific 4C-seq interrogation of the mouse Sox2 locus in ESCs with different deletions of regulatory regions. Overall: 4 baits; 14 different genotypes; 1-4 replicates