Functional maps of a genomic locus reveal confinement of an enhancer by its target gene [Region-capture Micro-C (RCMC)]

Genes are often activated by enhancers located at large genomic distances. The importance of this positioning is poorly understood. Here, by relocating different promoter-reporter constructs into >1,000 alternative positions within a single locus, we dissected the positional relationship between the mouse Sox2 gene and its distal enhancer. This revealed an intricate, sharply confined activation landscape, in which the native Sox2 gene occupies an optimal position for its activation. Deletion of the gene relaxes this confinement and broadly increases reporter activity. Surprisingly, this confining effect of the Sox2 gene on reporter activity is partially conferred by its ~1 kb coding region. Our local relocation approach provides high-resolution functional maps of a genomic locus and reveals that a gene can strongly constrain its enhancer's realm of influence. Overall design: Allele specific micro-C with capture of the Sox2 locus in different mouse embryonic stem cell lines containing a Sox2 reporter at different genomic locations in the presenc and absence of the endogenous Sox2::mCherry (S129) allele.