Multipartite super-enhancers function in an orientation-dependent manner

Abstract

Transcriptional enhancers regulate gene expression in a developmental-stage and cell-specific manner. They were originally defined as individual regulatory elements that activate expression regardless of distance and orientation to their cognate genes. Genome-wide studies have shown that the mammalian enhancer landscape is much more complex, with different classes of individual enhancers and clusters of enhancer-like elements combining in additive, synergistic and redundant manners, possibly acting as single, integrated regulatory elements. These so-called super-enhancers are largely defined as clusters of enhancer-like elements which recruit particularly high levels of Mediator and often drive high levels of expression of key lineage-specific genes. Here, we analysed 78 erythroid-specific super-enhancers and showed that, as units, they preferentially interact in a directional manner, to drive expression of their cognate genes. Using the well characterised α -globin super-enhancer, we show that inverting this entire structure severely downregulates α -globin expression and activates flanking genes 5' of the super-enhancer. Our detailed genetic dissection of the α -globin locus clearly attributes the cluster's functional directionality to its sequence orientation, demonstrating that, unlike regular enhancers, super-enhancers act in an orientation-dependent manner. Together, these findings identify a novel emergent property of super-enhancers and revise current models by which enhancers are thought to contact and activate their cognate genes.

Understanding fundamental principles of enhancer biology at a model locus

“…There are relatively few reports challenging the orientation of single enhancers in their natural chromatin context. [ 92–94 ] We have inverted R2, the major enhancer element in the α‐globin enhancer cluster, both in human and mouse loci in erythroid cultures and showed no effect on the expression of α‐globin, [ 92,94 ] supporting a single‐enhancer orientation‐independent function. However, some reports highlight the promiscuity of enhancers and their ability to simultaneously control more than one gene including genes lying upstream and downstream of the enhancer.…”

“…We have recently analysed the direction of interaction and the effects on gene expression of the cluster of α-globin enhancers. [92] To examine any effect of the orientation of enhancer clusters on transcription, we used the mouse α-globin locus as an experimental model.…”

“…The inverted SE interacts more with Rhbdf1 and Snrnp25, the genes lying 5′ of the native locus, in the direction of the inversion, upregulating both genes, as schematically shown and as reported in ref. [92] (C) Potential confounding factors, the CTCF boundary elements and the intervening MpG gene, were deleted. The phenotype persisted, demonstrating that the functional polarity is enhancer-cluster driven.…”

“…We have recently analysed the direction of interaction and the effects on gene expression of the cluster of α‐globin enhancers. [ 92 ] To examine any effect of the orientation of enhancer clusters on transcription, we used the mouse α‐globin locus as an experimental model. We have previously shown that the cluster of enhancers regulating α‐globin expression represents one of the most highly ranked SEs in erythroid cells [ 14 ] .…”

Experimental Validation and Prediction of Super-Enhancers: Advances and Challenges

“…Promoters of variable-expression genes lack these motifs and respond more strongly to enhancers [ 79 ]. Additionally, dissection/reinsertion experiments with well-characterized SEs, such as the erythroid α-globin SE [ 80 , 81 ], would aid in revising current models by which SEs are thought to contact and activate their cognate genes.…”