An Ancient Transcriptional Hub Couples Developmentally Regulated Gene Expression with Metabolism during Erythropoiesis

The α-globin genes and their regulation by the α-globin superenhancer have been extensively characterised. An adjacent gene, Nprl3, contains 4 of the 5 α-globin enhancers in mice, and 3 of 4 in humans. The neighbouring genomic positions of Nprl3 and α-globin, which share a topologically associated domain, have been maintained for >500 million years. However, the potential functional requirement of this genomic linkage has not been investigated until now. Nprl3 is conserved across Animalia and Fungi, and has been characterised to provide negative regulation of mTORC1, a critical controller of cellular metabolism. Here we report phenotypes of Nprl3-deficient erythropoiesis in mouse fetal liver and bone marrow, and in human peripheral blood. We provide molecular mechanistic insight that Nprl3 is essential for optimal erythropoiesis and for responding to fluctuating nutrient and growth factor concentrations. Finally, we show that erythroid Nprl3 function depends on transcriptional support arising from the α-globin enhancers.

Analysis of Nprl3^-/- fetal livers revealed severe impairment of erythroid development. On day E13.5, erythroid cells fail to develop beyond the proerythroblast stage. Loss of Nprl3 is accompanied by elevated mTORC1 signalling, confirming the canonical signalling role of Nprl3 in erythroblasts. Active mTORC1 typically inhibits autophagy; in consistence with this, we observed suppressed autophagic activity in Nprl3^-/- erythroblasts. We next investigated whether the erythroid defect results from haematopoietic-intrinsic Nprl3 requirements. This was tested using competitive bone marrow - fetal liver chimera experiments, in which Nprl3^-/- fetal liver cells contributed poorly to erythroid lineage reconstitution of irradiated adult bone marrow. Thus, Nprl3 regulates baseline erythropoiesis in the mouse.

To discover whether NPRL3 is also required for human erythropoiesis, NPRL3-knockout was induced in primary human CD34+ progenitors using a CRISPR-Cas9 RNP-editing system. These progenitors had reduced ability to produce enucleated erythroid cells compared to their negative control counterparts. NPRL3-knockout erythroblasts also demonstrated defective mTORC1 signalling responses to iron deficiency, amino acid withdrawal and erythropoietin stimulation. These results indicate that NPRL3 has a critical role in interpreting fluctuating nutritional environments, and in tuning the metabolic response of developing erythroid cells to the extracellular milieu.

Importantly, Nprl3 expression is known to be elevated in erythroid cells, increasing ~30-fold during erythroid lineage commitment due to α-globin enhancer activity. We sought to discover whether this erythroid-specific increase in Nprl3 expression is required for the functionality outlined above. Using genetic engineering to delete the Nprl3 promoter on one allele, and to delete all α-globin enhancers on the other, we eliminated all interactions between Nprl3 and α-globin regulatory elements. Embryos heterozygous for both alleles (Nprl3^+/-α-globin-enhancers^+/-,'Nalph') retain non-erythroid 'un-enhanced' Nprl3 expression levels, and enhancer-regulated α-globin expression. Remarkably, at E13.5, the Nalph genotype presented impaired erythropoiesis reminiscent of that observed in Nprl3^-/- embryos, with development inhibited at the same stage of differentiation. Thus, we conclude that the genomic contact hub Nprl3 shares with α-globin and its enhancers bestows the erythroid-specific transcriptional upregulation required for Nprl3 to perform its important erythropoietic role. This finding suggests that the deep evolutionary coupling of these two genes has enabled the α-globin enhancers to control metabolism, as well as α-globin production, in developing erythroid cells.

Davies:Nucleome: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company. Drakesmith:Pfizer / Imara / Keros: Consultancy; Procter and Gamble: Research Funding; Pharmacosmos / Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees.