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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.

In the last few years, the advent of new technological approaches has led to a better knowledge of the ontogeny of erythropoiesis during development and of the journey leading from hematopoietic stem cells (HSCs) to mature red blood cells (RBCs). Our view of a well-defined hierarchical model of hematopoiesis with a near-homogeneous HSC population residing at the apex has been progressively challenged in favor of a landscape where HSCs themselves are highly heterogeneous and lineages separate earlier than previously thought. The coordination of these events is orchestrated by transcription factors (TFs) that work in a combinatorial manner to activate and/or repress their target genes. The development of next generation sequencing (NGS) has facilitated the identification of pathological mutations involving TFs underlying hematological defects. The examples of GATA1 and KLF1 presented in this review suggest that in the next few years the number of TF mutations associated with dyserythropoietic disorders will further increase.

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.

Prospective 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.

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.

Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al., 2005).Cells can be driven into granulocytic/neutrophil differentiation through the use of high doses of retinoic acid (RA), which overcomes the differentiation block. It should be noted that these pharmacological doses of RA are not compatible with erythroid differentiation, and it is hence not viable to obtain robust erythroid and myeloid differentiation in the same assay. Indeed, colonies scored as mixed-lineage in CFC assays are a mixture of undifferentiated and erythroid cells (Tsai et al., 1994). Nevertheless, robust single-lineage erythroid and neutrophil differentiation can be obtained in liquid culture under defined cytokine conditions, as specified below.