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Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.

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Background Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed. Methods In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin. Results We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts. Conclusion As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies. Electronic supplementary material The online version of this article (10.1186/s13287-019-1231-z) contains supplementary material, which is available to authorized users.

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle‐cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR‐mediated genome editing of an immortalised human erythroblast cell line (BEL‐A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Fynull), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof‐of‐principle demonstration of combinatorial CRISPR‐mediated blood group gene editing to generate customisable or multi‐compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.