Bystander activation across a TAD boundary supports a cohesin-dependent hub-model for enhancer function

Enhancers in the mammalian genome are able to control their target genes over very large genomic distances, often across intervening genes. Yet the spatial and temporal specificity of developmental gene regulation would seem to demand that enhancers are constrained so that they only activate the correct target gene. The sculpting of three-dimensional chromosome organization, especially that brought about through cohesin-dependent loop extrusion, is thought to be important for facilitating and constraining the action of enhancers. In particular, the boundaries of topologically associating domains (TADs) are thought to delimit regulatory landscapes and prevent enhancers acting on genes close in the linear genome, but located in adjacent TADs. However, there are some examples where enhancers appear to act across TAD boundaries. In these cases it was not determined whether an enhancer can simultaneously activate transcription at genes in its own TAD and in an adjacent TAD. Here, using a combination of mouse developmental genetics, and synthetic activators in stem cells, we show that some Shh enhancers can activate transcription simultaneously, not only of Shh but also at a gene Mnx1 located in an adjacent TAD. This occurs in the context of a chromatin configuration that maintains both genes and the enhancers close together and is influenced by cohesin. To the best of our knowledge this is the first report of two endogenous mammalian genes transcribed simultanously under the control of the same enhancer, and across a TAD boundary. Our data have implications for understanding the design rules of gene regulatory landscapes, and are most consistent with a transcription hub model of enhancer-promoter communication.