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Although establishment and maintenance of mitochondria are essential for the production of massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells (RBCs), thus creating a biphasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondrial retention in RBCs, suggesting that a proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Because the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron or heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and the induction of mitophagy. GATA1 might induce the expression of the autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a biphasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondrial disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1−/− accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1−/− (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

The hematopoietic transcription factor GATA1 induces heme accumulation during erythropoiesis by directly activating genes mediating heme biosynthesis. In addition to its canonical functions as a hemoglobin prosthetic group and enzyme cofactor, heme regulates gene expression in erythroid cells both transcriptionally and post-transcriptionally. Heme binding to the transcriptional repressor BACH1 triggers its proteolytic degradation. In heme-deficient cells, BACH1 accumulates and represses transcription of target genes, including α- and β-like globin genes, preventing the accumulation of cytotoxic free globin chains. A recently described BACH1-independent mechanism of heme-dependent transcriptional regulation is associated with a DNA motif termed heme-regulated motif (HERM), which resides at the majority of loci harboring heme-regulated chromatin accessibility sites. Progress on these problems has led to a paradigm in which cell type-specific transcriptional mechanisms determine the expression of enzymes mediating the synthesis of small molecules, which generate feedback loops, converging upon the transcription factor itself and the genome. This marriage between transcription factors and the small molecules that they control is predicted to be a canonical attribute of regulatory networks governing cell state transitions such as differentiation in the hematopoietic system and more broadly.

Developmental-regulatory networks often include large gene families encoding mechanistically-related proteins like G-protein-coupled receptors, zinc finger transcription factors and solute carrier (SLC) transporters. In principle, a common mechanism may confer expression of multiple members integral to a developmental process, or diverse mechanisms may be deployed. Using genetic complementation and enhancer-mutant systems, we analyzed the 456 member SLC family that establishes the small molecule constitution of cells. This analysis identified SLC gene cohorts regulated by GATA1 and/or GATA2 during erythroid differentiation. As >50 SLC genes shared GATA factor regulation, a common mechanism established multiple members of this family. These genes included Slc29a1 encoding an equilibrative nucleoside transporter (Slc29a1/ENT1) that utilizes adenosine as a preferred substrate. Slc29a1 promoted erythroblast survival and differentiation ex vivo. Targeted ablation of murine Slc29a1 in erythroblasts attenuated erythropoiesis and erythrocyte regeneration in response to acute anemia. Our results reveal a GATA factor-regulated SLC ensemble, with a nucleoside transporter component that promotes erythropoiesis and prevents anemia, and establish a mechanistic link between GATA factor and adenosine mechanisms. We propose that integration of the GATA factor-adenosine circuit with other components of the GATA factor-regulated SLC ensemble establishes the small molecule repertoire required for progenitor cells to efficiently generate erythrocytes., Author summary GATA transcription factors endow blood stem and progenitor cells with activities to produce progeny that transport oxygen to protect cells and tissues, evade pathogens and control physiological processes. GATA factors regulate hundreds of genes, and the actions of these genes mediate important biological functions. While the genes have been documented, many questions remain regarding how the “network” components mediate biological functions. The networks include members of large gene families, and the relationships between the regulation and function of individual family members is not well understood. Analyzing datasets from genetic complementation and enhancer mutant systems revealed that GATA factors regulate an ensemble of membrane transporters termed solute carrier proteins (SLCs), which dictate the small molecule composition of cells. Genetic analyses with Slc29a1, which transports adenosine, revealed its function to promote erythrocyte development, and Slc29a1 attenuated anemia in a mouse model. This study revealed the importance of SLC transporters in GATA factor networks. We propose that the GATA factor-adenosine circuit integrates with other SLCs to establish/maintain the small molecule constitution of progenitor cells as a new mechanism to control blood cell development.

By functioning as an enzyme cofactor, hemoglobin component and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we used CRISPR/Cas9 to delete intron 1 and 8 GATA motifs in Alas2, encoding the rate-limiting enzyme in heme biosynthesis, in erythroid precursor G1E-ER-GATA-1 cells (Tanimura et al., 2016, EMBO Rep.). These cells stably express a b-estradiol-activated allele encoding the estrogen receptor hormone binding domain fused to GATA-1. The GATA motif mutations abrogated GATA-1-mediated activation of Alas2 and decreased heme ~30 fold. We merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Slc30a1 and the zinc importer Slc39a8 were the most highly expressed zinc transporters in G1E-ER-GATA-1 cells. Both were GATA-1-activated, and Slc30a1, but not Slc39a8, expression declined in heme-deficient Alas2-intronic mutant cells. We tested whether the zinc transporter genes Slc30a1 and Slc39a8 that share GATA-1 regulation and differ in heme regulation are controlled similarly in primary cells. Lineage-depleted hematopoietic precursors from murine fetal livers were cultured for 72 hours, and flow cytometry was used to isolate cells based on erythroid surface markers. Whereas Slc30a1 expression increased 5.7-fold upon differentiation, Slc39a8 increased 4.6-fold during the initial phase, but was downregulated 8.7-fold thereafter. To quantify intracellular zinc levels, we used the fluorescent zinc indicator FluoZin-3. The FluoZin-3 signal increased during early maturation and decreased thereafter, as predicted by the zinc transporter switch, in which expression of the zinc exporter and importer was sustained and decreased, respectively, during terminal differentiation. We demonstrated that the zinc transporter switch occurs in a primary human erythroblast culture system. Intracellular zinc rises during initial erythroid maturation, followed by a steep decline during terminal differentiation. The heme biosynthesis inhibitor succinylacetone reduced SLC30A1 expression 2.8-fold (p < 0.01), as well as other heme-dependent globin genes HBB and HBA1. These primary mouse and human cell studies revealed an evolutionarily conserved mechanism to differentially regulate zinc transporter genes during erythroid differentiation, culminating in a switch from importer and exporter expression to solely exporter expression. To test whether reconfiguring the mechanism governing intracellular zinc impacts differentiation and to elucidate Slc30a1 and Slc39a8 function, we developed multiple shRNAs and used them in loss-of-function studies with lineage-negative hematopoietic precursors from murine fetal livers (E14.5). Slc39a8 downregulation decreased intracellular zinc levels in immature erythroblasts. We also used the zinc chelator TPEN to reduce zinc in immature erythroblasts. TPEN reduced intracellular zinc and considerably decreased the live cell population (p < 0.001). Downregulating Slc30a1 increased intracellular zinc 2.2-fold and, strikingly, accelerated differentiation (p < 0.001). This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation. Ongoing studies involve the development of innovative strategies to leverage this mechanism to enhance or suppress erythrocyte development with primary mouse and human systems. We are conducting multi-disciplinary metallomic analyses to test the hypothesis that the dynamic changes in zinc selectively commission and decommission zinc-binding proteins in erythroblasts. We anticipate that this approach can be readily extended to other sectors of the hematopoietic system and more broadly. Disclosures No relevant conflicts of interest to declare.