Graphical AbstractHighlights d The structure of the mouse a-globin locus is investigated with polymer physics models d The conformation is highly intermingled when the globin genes are inactive in ESCs d The locus is folded in a hairpin-shaped conformation when active in erythroid cells d The model accurately predicts three-way contacts, as confirmed by TriC experimental data Correspondence chiariello@na.infn.it (A.M.C.), mario.nicodemi@na.infn.it (M.N.) In BriefChiariello et al. use polymer physics models to infer the 3D conformations of the murine a-globin locus. In the transition from ESCs to erythroid cells, the locus rearranges from an inactive highly intermingled conformation to an active, hairpin-shaped structure, marked by cell-specific three-way contacts accurately predicted by the models. SUMMARYWe investigate the three-dimensional (3D) conformations of the a-globin locus at the single-allele level in murine embryonic stem cells (ESCs) and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent fluorescence in situ hybridization (FISH) data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ESCs to erythroid cells, associated with the activation of the globin genes. We find that in ESCs, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, whereas in erythroid cells the a-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific geneenhancer contacts, suggesting that beyond the interaction of CTCF sites, multiple molecular mechanisms cooperate to form an interacting domain.

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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.