Expression of the erythroid controller of systemic iron homeostasis erythroferrone is cis-regulated

Abstract Erythroferrone (ERFE) is secreted from erythroblasts in response to increased erythropoiesis to suppress hepcidin, enabling enhanced nutritional iron absorption and iron release from the reticuloendothelial system, facilitating iron availability to the bone marrow and hence haemoglobin and thus red cell production1. In erythropoietic disorders such as thalassaemia, ineffective erythropoiesis results in erythroferrone upregulation, chronic hepcidin suppression and iron overload2. Despite the critical role of erythroferrone in systemic iron homeostasis and an emerging role as a therapeutic target, the molecular regulation of ERFE expression has not been described. Here, we sought to characterize the genomic regulation of ERFE using a human erythroblast model. We leveraged a well-characterized conditionally immortalized EPO-dependent human erythroid progenitor cell model (HUDEP-2) and conducted in vitro erythroid differentiation to generate late-stage orthochromatic erythroblasts. First, we confirmed that ERFE mRNA expression is increased at the intermediate stage of erythroblast differentiation in human-derived cells, consistent with previous animal data1. Next, we deployed ATAC-Seq and CUT&RUN techniques to identify accessible chromatin and characterise the chromatin modification landscape around the ERFE locus. We first sorted erythroblast stages and applied ATAC-Seq to each stage. This demonstrated a region of accessible chromatin 3' of the coding sequence of ERFE, located within the gene's 3' Untranslated Region (UTR). In pro-erythroblasts we detected a single peak, which expanded to four distinct peaks by the late erythroblast stage and was also marked by enhancer-related H3K4me1 and H3K27ac chromatin marks. Next, we undertook CUT&RUN experiments across erythroid maturation and demonstrated that canonical erythroid transcription factors GATA1, KLF1 and TAL1 bind within the ATAC-Seq peaks of the putative 3' UTR enhancer region in a cell-stage specific pattern. We undertook chromosome conformation capture (Capture-C) and identified interactions between the ERFE promotor region and the 3'UTR putative enhancer region. Finally, we confirmed the functional importance of this region in the regulation of ERFE by using CRISPR-Cas9 to create indels in the ATAC-Seq peak that was observed to be present in pro-erythroblasts. Two biallelic deletions were generated, ERFE-KO1 (66bp deletion) and ERFE-KO2 (25bp deletion). ERFE mRNA expression was compared between the knockout lines and a non-targeting sgRNA control line. At the pro-erythroblast stage, we observed a 113-fold (ERFE-KO1) and 12-fold reduction (ERFE-KO2) in ERFE mRNA expression relative to the non-targeted control, while at the intermediate erythroblast stage, we observed an 11-fold and 6-fold reduction in ERFE expression, respectively. These are the first data describing the genomic regulation of ERFE and highlight a critical role for cis-regulation in the gene's activation. References 1. Kautz L, Jung G, Valore EV, Rivella S, Nemeth E, Ganz T. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014;46(7):678-684. 2. Kautz L, Jung G, Du X, et al. Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of β-thalassemia. Blood. 2015;126(17):2031-2037.