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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.

The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6−/− red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562 cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation. To obtain an overview on processes downstream to Sox6 expression, we performed a differential proteomic analysis on human erythroid K562 cells overexpressing Sox6. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24 A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these proteins during erythropoiesis.

SOX6 is a HMG-box transcription factor expressed in a wide range of tissues. Recent data show that SOX6 expression is altered in different cancers, in the majority of cases being downregulated. To date, no data are available about SOX6 role in hematological malignancies. Here we demonstrate that SOX6 overexpressing BCR-ABL1+ B-ALL cells are unable to promote leukemia in a mouse model. Starting from this observation, we extended our study to a panel of human leukemic cells carrying genetic lesions distinctive of different types of leukemias and myeloproliferative disorders (the BCR-ABL1 translocation and the JAK2V617F amino acid substitution) to dissect the cellular events induced by SOX6. The inhibition of proliferation is the invariant outcome of SOX6 overexpression but it is achieved via two different cellular responses: terminal differentiation in erythroid-biased cells, irrespectively of their mutation, and apoptosis in megakaryocytic-primed and lymphoid cells. Within this context, cells carrying the highest copy number of the JAK2V617F allele better counteract the SOX6-imposed growth arrest. The interrogation of the GEPIA (Gene Expression Profiling Interactive Analysis) human dataset reveals that SOX6 is downregulated in a cohort of AML patients, uncovering a wide anti-proliferative role of SOX6 in a variety of mutant backgrounds.

SOX6 is a HMG-box transcription factor expressed in a wide range of tissues. Recent data show that SOX6 expression is altered in different cancers, in the majority of cases being downregulated. To date, no data are available about SOX6 role in hematological malignancies. Here we demonstrate that SOX6 overexpressing BCR-ABL1+ B-ALL cells are unable to promote leukemia in a mouse model. Starting from this observation, we extended our study to a panel of human leukemic cells carrying genetic lesions distinctive of different types of leukemias and myeloproliferative disorders (the BCR-ABL1 translocation and the JAK2V617F amino acid substitution) to dissect the cellular events induced by SOX6. The inhibition of proliferation is the invariant outcome of SOX6 overexpression but it is achieved via two different cellular responses: terminal differentiation in erythroid-biased cells, irrespectively of their mutation, and apoptosis in megakaryocytic-primed and lymphoid cells. Within this context, cells carrying the highest copy number of the JAK2V617F allele better counteract the SOX6-imposed growth arrest. The interrogation of the GEPIA (Gene Expression Profiling Interactive Analysis) human dataset reveals that SOX6 is downregulated in a cohort of AML patients, uncovering a wide anti-proliferative role of SOX6 in a variety of mutant backgrounds.

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.

Transcriptional regulation is a key step in the commitment and differentiation of hemopoietic cells. This process is achieved by a network of interacting lineage-specific transcription factors acting in conjunction with more-general transcription factors. GATA-1 is a zinc finger transcription factor expressed within the hemopoietic system, in the erythroid and megakaryocytic lineages, and in mast cells and eosinophils. Knockout studies revealed that GATA-1 is required for the normal maturation of erythroid and megakaryocytic cells. Consistent with this, functional GATA-1 elements are known to regulate the expression of virtually all erythroid and megakaryocytic genes studied, including transcription factors such as EKLF and p45 NF-E2, which in turn regulate important aspects of the specification of these lineages. The transcriptional activity of GATA-1 is modulated by a complex network of directly interacting proteins, including FOG1, LMO-2, EKLF, p300, and Pu1; depending on the partner and on the cellular context, the outcome of these interactions can be synergism or cross-antagonism (5, 11, 32). CP2 is a ubiquitously expressed transcription factor belonging to the Drosophila grainyhead-like gene family. CP2 consists of homo/heterodimers of several protein isoforms produced by alternative splicing, from two different genomic loci, LBP-1c and LBP-1a (the human homologs of mouse CP2c and CP2a). CP2 binds as a dimer to a CNRG (N5-6)CNRG DNA motif, present in diverse cellular and viral promoters (23, 48, 50). CP2c was originally identified by its ability to stimulate the transcription of the α-globin gene (19); it appears to be involved in the fetal erythroid expression of the γ-globin gene through the formation of a heterodimer with the erythroid transcription partner NF-E4 (the stage selector protein) (14, 51). Indeed, in transgenic experiments, the mutation of the stage selector protein binding site on the γ-globin promoter (the stage selector element [SSE]) shows that this region affects the γ- versus β-globin ratio of expression in early stages of fetal development (34). Moreover, overexpression of antisense CP2 mRNA in MEL cells undergoing erythroid differentiation in vitro not only suppresses α-globin expression but also impairs β-globin expression and hemoglobinization (8), suggesting that CP2 binding to the promoter is essential for optimal globin transcription in erythroid cells (7). The transcriptional regulation of GATA-1 is coordinated by several regulatory elements located both 5′ to the transcriptional start site and in the first intron (25, 29, 30, 35, 39, 45). The region upstream of the erythroid promoter, also reported as hypersensitivity site 2 (HS2), contains a double GATA motif shown to be essential for the erythroid promoter activity (28, 42, 44). This sequence, foot printed in vivo in erythroid cells, represents a strong double GATA-1 binding site, which has been proposed to mediate autoregulation by GATA-1 itself. Mice harboring a 21-base-pair deletion in this motif in the endogenous GATA-1 locus (49) show a lack of eosinophil production, while platelets and mast cells appear normal. Erythropoiesis is also abnormal in these animals, which display a reduction in red cell number, hematocrit, and hemoglobin. The common role of GATA-1 and CP2 in regulating erythroid genes suggested the possibility that this could be occurring cooperatively. Here, we show that this is true for GATA-1 gene transcription, in which the two factors act through adjacent binding sites present on the HS2 erythroid enhancer element. We show that CP2 binds to HS2, creating a ternary complex with GATA-1 and DNA, and that mutations in the CP2 consensus greatly impair HS2 activity in transient transfection assays with K562 cells. Adjacent GATA-1 and CP2 binding sites are also present in regulatory elements of several other genes expressed in the hematopoietic lineage. Among them, we show that a CP2 binding site adjacent to functionally relevant GATA-1 sites is important for the activities of the p45NF-E2 and EKLF promoters. Chromatin immunoprecipitation (ChIP) experiments reveal that CP2 is bound in vivo to the regulatory elements of the GATA-1, EKLF, and p45 NF-E2 genes and that at least for the first two genes, it is bound simultaneously to GATA-1. Finally, GATA-1 and CP2 can physically interact, even in the absence of DNA, as demonstrated by immunoprecipitation and glutathione S-transferase (GST) pull-down experiments. Taken together, these data suggest that adjacent GATA-1 and CP2 sites contribute to the regulation of the GATA-1, p45 NF-E2, and EKLF genes.

SummaryProspective isolation is critical for understanding the cellular and molecular aspects of stem cell heterogeneity. Here, we identify the cell surface antigen CD9 as a positive marker that provides a simple alternative for hematopoietic stem cell isolation at high purity. Crucially, CD9 affords the capture of all hematopoietic stem cells in murine bone marrow in the absence of contaminating populations that lack authentic stem cell function. Using CD9 as a tool to subdivide hematopoietic stem-cell-containing populations, we provide evidence for heterogeneity at the cellular, functional, and molecular levels.

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.