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

Mammalian enhancers can regulate genes over large genomic distances, often skipping over other genes. Despite this, precise developmental regulation suggests that mechanisms exist to ensure enhancers only activate their correct targets. Sculpting of three-dimensional chromosome organization through cohesin-dependent loop extrusion is thought to be important for facilitating and constraining enhancer action. The boundaries of topologically associating domains (TADs) are thought to prevent enhancers acting on genes in adjacent TADs. However, there are examples where enhancers appear to act across TAD boundaries, but it has remained unclear whether a single enhancer can simultaneously activate genes in different TADs. Here we show that some Shh enhancers can activate transcription concurrently not only at <i>Shh</i> but also at <i>Mnx1</i> located in an adjacent TAD. This occurs in the context of a chromatin conformation maintaining genes and enhancers in close proximity and is influenced by cohesin. To our knowledge, this is the first report of two endogenous mammalian genes transcribed concurrently under the control of the same enhancer and across a TAD boundary. These findings have implications for understanding the design rules of gene regulatory landscapes and are consistent with a transcription cluster model of enhancer-promoter communication.