Super-enhancers (SEs) are a class of compound regulatory elements which control expression of key cell-identity genes. It remains unclear whether they are simply clusters of independent classical enhancer elements or whether SEs manifest emergent properties and should therefore be considered as a distinct class of element. Here, using synthetic biology and genome editing, we engineered the well characterised erythroid α-globin SE at the endogenous α-globin locus, removing all SE constituents in a mouse embryonic stem cell-line, to create a "blank canvass". This has allowed us to re-build the SE through individual and combinatorial reinsertion of its five elements (R1, R2, R3, Rm, R4), to test the importance of each constituent's sequence and position within the locus. Each re-inserted element independently creates a region of open chromatin and binds its normal repertoire of transcription factors; however, we found a high degree of functional interdependence between the five constituents. Surprisingly, the two strongest α-globin enhancers (R1 and R2) act sub-optimally both on their own and in combination, and although the other three elements (R3, Rm and R4) exhibit no discernible enhancer activity, they each exert a major positive effect in facilitating the activity of the classical enhancers (R1 and R2). This effect depends not simply on the sequence of each elements but on their positions within the cluster. We propose that these "facilitators" are a novel form of regulatory element, important for ensuring the full activity of SEs but are distinct from conventional enhancer elements.

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.