Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation

Abstract

Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell’s surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

Regulation of GATA1 levels in erythropoiesis

“…Interestingly, mutations in the RPS14 gene were also found in patients with primary myelofibrosis and were again associated with reduced GATA1 protein levels and impaired megakaryopoiesis, thus linking RP gene mutations and reduced GATA1 protein levels in hematological disease affecting a hematopoietic lineage other than the erythroid lineage. Furthermore, it was recently shown that heme levels are retroactively involved in this spectrum of phenomena around RP dependent GATA1 protein level regulation . This study showed that in early erythroid progenitors, heme induces the translation of RPs, ensuring sustained globin synthesis.…”

“…A subsequent study showed a disequilibrium of globin chain and heme synthesis in RPL11‐ and RPL5‐deficient human erythroid cells, resulting in excessive free heme which downregulated HSP70 protein . The study by Doty et al also suggests that aberrant accumulation of heme, contributes to the amplification of RP imbalance, thus causing premature downregulation of GATA1 levels, responsible for the IE that is characteristic of DBA and MDS . Hence, it is clear in that in DBA, HSP70 downregulation results in a significant drop in GATA1 protein levels due to caspase‐mediated cleavage, which underlies the observed erythroid maturation defects .…”

“…This study showed that in early erythroid progenitors, heme induces the translation of RPs, ensuring sustained globin synthesis. However, at later stages of differentiation, heme accumulation associates with downregulation of Gata1 through a still undescribed mechanism …”

GATA1 mutations in red cell disorders

“…Two recent papers suggest that decrease of GATA1 full-length protein resulting from RP haploinsufficiency and deficiency of HSP70 can disturb the balance of globin-heme and leads to the accumulation of free cytoplasmic heme in erythroid progenitors, which can cause increase of P53-dependent apoptosis of DBA erythroid cells. [119][120][121] Of note, unlike in the presence of RP mutations in DBA patients, accumulation of heme upon only FLVCR1 depletion causes P53-independent apoptosis. 120 Finally, work by Bodine and colleagues suggest yet another model for pathogenesis of DBA.…”

“…[119][120][121] Of note, unlike in the presence of RP mutations in DBA patients, accumulation of heme upon only FLVCR1 depletion causes P53-independent apoptosis. 120 Finally, work by Bodine and colleagues suggest yet another model for pathogenesis of DBA. 122 By culturing CD34 + cells from patients, they found that both RP and GATA1 mutant cells showed reduced proliferation and delayed differentiation.…”