Genomic context mediates position-dependent enhancer function at the Sonic hedgehog locus

Enhancers can regulate their target promoters over large genomic distances for which spatial proximity between these elements is assumed to be necessary. Topologically Associating Domains (TADs) are thought to provide such a spatial environment for regulatory domains harboring multiple enhancers driving complex expression of developmental genes. However, how enhancer position within the TAD affects its function in vivo is not known. Here, we address this question by generating a series of genome-engineered mouse lines in which we systematically reposition the limb enhancer of Sonic hedgehog (Shh), ZRS, to various positions within and outside of its TAD to analyze positional effects on gene expression, chromatin folding, and limb phenotype. We find that the enhancer activates Shh only when located within the same TAD, however, its activity varies across intra-TAD positions. ZRS-driven Shh expression drops rapidly with increasing distance from the promoter but eventually plateaus irrespective of linear distance. Moreover, engineering of 3D enhancer-promoter contacts through integration of different CTCF sites reveals that increased contact frequencies do not directly translate into increased transcriptional output and may cause undesired consequences in vivo due to detrimental effects on Shh expression in other tissues. Finally, we show a selective ability of the ZRS enhancer to activate non-target genes in ectopic TADs. Our work highlights the importance of genomic context within TADs for enhancer function and our limited understanding of the interplay between regulatory sequences within these domains. Capture Hi-C experiments from wildtype and mutant E10.5 mouse limb buds