Multipartite super-enhancers function in an orientation-dependent manner [ChIP-Seq]

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 a-globin super-enhancer, we show that inverting this entire structure severely downregulates a-globin expression and activates flanking genes 5’ of the super-enhancer. Our detailed genetic dissection of the a-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. An inversion spanning the alpha globin superenhancer and including both the Nprl3 and Mpg genes was genetically engineered in mouse ESCs and a mouse model was created by blastocyst injection of these mESCs. Primary erythroid cells derived from APH-treated spleens of these mice (INVEN) as well as WT mice were subjected to various analyses (ATAC-Seq, ChIP-Seq, RNA-Seq, Capture C) to examine the effect this inversion has on the expression of the erythroid -specific alpha globin genes. Embryonic blood (E10.5) was also analysed to differentiate the effect of the inversion on primitive (embryonic) versus dfinitive (adult) erythropoiesis. Moues ESCs were also generated; these harbour additional mutations that allow the effect of the inversion to be untangled from other confoudning factors (such as the boundary elements HS3839 or the repoistioned Mpg gene). These mESCs were subjected to an in vitro erythroid differentiation protocol (doi: 10.1371/journal.pone.0261950) and purified CD71+ erythroid cells derived from differentiated Wildtype (WT) or mutant mouse Embryonic Stem (mES) cells were analysed similarly to the primary spleen erythroid cells.