Endogenous small molecule effectors in GATA transcription factor mechanisms governing biological and pathological processes.

Transcriptional mechanisms establish and maintain complex genetic and protein networks to control cell state transitions. The hematopoietic transcription factor GATA1 is a master regulator of erythropoiesis and megakaryopoiesis, and human GATA1genetic variants cause anemia and megakaryoblastic leukemia. Multiomic analyses revealed that GATA1 controls expression of transporters and metabolic enzymes that dictate intracellular levels of endogenous small molecules, including heme, metal ions, and sphingolipids. Besides its canonical function as a hemoglobin component, heme facilitates or antagonizes GATA1 function to regulate erythropoiesis via mechanisms dependent or independent of the heme-binding transcription factor BTB domain and CNC homology 1 (BACH1). GATA1 regulates the expression of genes encoding heme biosynthetic enzymes and BACH1. GATA1 maintains homeostasis of bioactive ceramides during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Disrupting ceramide homeostasis impairs critical cytokine signaling and is detrimental to erythroid cells. During erythroid maturation, GATA1 induces a zinc transporter switch that favors export versus import, thus dictating the intracellular zinc level, erythroblast survival, and differentiation. In aggregate, these studies support an emerging paradigm in which GATA factor-dependent transcriptional mechanisms control the intracellular levels of endogenous small molecules and small molecule-dependent feedback loops that serve as vital effectors of transcription factor activity, genome function, and cell state transitions.

A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process.

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

Heme-dependent induction of mitophagy program during differentiation of murine erythroid cells.

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.

Heme as a differentiation-regulatory transcriptional cofactor.

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 ß-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.

Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation.

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.

GATA factor-regulated solute carrier ensemble reveals a nucleoside transporter-dependent differentiation mechanism.

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.