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The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of β-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and β039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the β/γ+β globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the β-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

Summary We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs) enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2., Graphical Abstract, Highlights • We present a method for inferring gene regulatory networks (GRNs) from single cells • Lineage cross-antagonism is a key property of GRNs of early lineage commitment • Ddit3 is a regulatory node in erythroid lineage programming • A Ddit3-Gata2 regulatory axis antagonizes myeloid and enables erythroid programs, Pina et al. develop a gene regulatory network inference method using single-cell gene expression data and identify Ddit3 as a regulatory node in erythroid lineage programming. The authors explore this inference and show that Ddit3 can antagonize myeloid programming and enable erythroid signatures and forms a regulatory axis with Gata2.

The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein, we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2+/−) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin−CD117+Sca-1−CD34+CD16/32high granulocyte-macrophage progenitor (GMP) compartment of GATA-2+/− marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. Although apoptosis and cell-cycle distribution remained unperturbed in GATA-2+/− GMP, quiescent cells from GATA-2+/− GMP exhibited altered functionality. Gene expression analysis showed attenuated expression of HES-1 mRNA in GATA-2–deficient GMP. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3, and enforced expression of HES-1 expression in GATA-2+/− GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as a novel, pivotal determinant of GMP cell fate.

The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown. We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis. These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.

Sox6 belongs to the Sry (sex-determining region Y)–related high-mobility-group–box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.

Analysis of individual cells within phenotypically-uniform stem and progenitor populations reveals heterogeneity of cell states and affords exploration of their differential contribution to fate transitions. Herein, we investigate the transcriptional composition of lineage commitment transitions in the mouse hematopoietic system, obtained either spontaneously or driven by individual transcription factors (TF), to define alternative molecular states at the emergence of erythroid and myeloid lineage identity. To this end, we transcriptionally profile hundreds of individual cells across a spectrum of lineage states by quantitative RT-PCR on a Fluidigm platform. We explore the dual binary (on-off) vs. continuous (expression level) nature of PCR data and respectively calculate odds ratio, and Spearman correlation on co-expressing cells, for any pair of genes, in order to infer the circuitry underlying alternative routes to lineage commitment. This approach identifies Ddit3 as a specific target of GATA-driven networks that antagonizes Cebpa transcriptionally at the emergence of myeloid fate. Knockdown and knockout approaches indicate a requirement for Ddit3 in early erythroid decisions from stem cells. Enforced expression of Ddit3 in granulocytic-monocytic progenitors (GMP) results in down-regulation of individual Cebpfamily members and their downstream transcriptional programmes, including myelo-monocytic cytokine receptors. Notably, the resulting cells have robust self-renewal potential and express an early erythroid signature that is augmented in the presence of interleukin-3 and erythropoietin. Taken together, the data suggest that Ddit3 can nucleate incipient erythroid-affiliated programmes in early erythro-myelopoiesis, and may contribute to fate choice through antagonism with Cebp networks. Interestingly, ex-vivo culture and expression profiling of individual wild-type and DDIT3-enforced GMP suggest that the erythroid-primed state captured through Ddit3 expression may be found in a small proportion of native GMP, suggesting some fluidity of lineage identity in early progenitors. Disclosures: No relevant conflicts of interest to declare.