The Coup-TFII orphan nuclear receptor is an activator of the γ-globin gene.

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 ß-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 ß039 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 ß/γ+ß globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the ß-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

Defective erythroid maturation in gelsolin mutant mice.

BACKGROUND: During late differentiation, erythroid cells undergo profound changes involving actin filament remodeling. One of the proteins controlling actin dynamics is gelsolin, a calcium-activated actin filament severing and capping protein. Gelsolin-null (Gsn(-/-)) mice generated in a C57BL/6 background are viable and fertile.1 DESIGN AND METHODS: We analyzed the functional roles of gelsolin in erythropoiesis by: (i) evaluating gelsolin expression in murine fetal liver cells at different stages of erythroid differentiation (using reverse transcription polymerase chain reaction analysis and immunohistochemistry), and (ii) characterizing embryonic and adult erythropoiesis in Gsn(-/-) BALB/c mice (morphology and erythroid cultures). RESULTS: In the context of a BALB/c background, the Gsn(-/-) mutation causes embryonic death. Gsn(-/-) embryos show defective erythroid maturation with persistence of circulating nucleated cells. The few Gsn(-/-) mice reaching adulthood fail to recover from phenylhydrazine-induced acute anemia, revealing an impaired response to stress erythropoiesis. In in vitro differentiation assays, E13.5 fetal liver Gsn(-/-) cells failed to undergo terminal maturation, a defect partially rescued by Cytochalasin D, and mimicked by administration of Jasplakinolide to the wild-type control samples. CONCLUSIONS: In BALB/c mice, gelsolin deficiency alters the equilibrium between erythrocyte actin polymerization and depolymerization, causing impaired terminal maturation. We suggest a non-redundant role for gelsolin in terminal erythroid differentiation, possibly contributing to the Gsn(-/-) mice lethality observed in mid-gestation.

Physiological and Aberrant γ-Globin Transcription During Development.

The expression of the fetal Gγ- and Aγ-globin genes in normal development is confined to the fetal period, where two γ-globin chains assemble with two α-globin chains to form α2γ2 tetramers (HbF). HbF sustains oxygen delivery to tissues until birth, when ß-globin replaces γ-globin, leading to the formation of α2ß2 tetramers (HbA). However, in different benign and pathological conditions, HbF is expressed in adult cells, as it happens in the hereditary persistence of fetal hemoglobin, in anemias and in some leukemias. The molecular basis of γ-globin differential expression in the fetus and of its inappropriate activation in adult cells is largely unknown, although in recent years, a few transcription factors involved in this process have been identified. The recent discovery that fetal cells can persist to adulthood and contribute to disease raises the possibility that postnatal γ-globin expression could, in some cases, represent the signature of the fetal cellular origin.

ß-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies.

Hemoglobin is a tetrameric protein composed of two α and two ß chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and ß-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two ß subunits (α2ß2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in ß-globin production cause two of the most common monogenic-inherited disorders: ß-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of ß-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.

The Pleiotropic Effects of GATA1 and KLF1 in Physiological Erythropoiesis and in Dyserythropoietic Disorders.

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.

SOX6 blocks the proliferation of BCR-ABL1+ and JAK2V617F+ leukemic cells.

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.

Unravelling pathways downstream Sox6 induction in K562 erythroid cells by proteomic analysis.

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.

A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds.

The identification of drugs capable of reactivating γ-globin to ameliorate ß-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/ß globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing ß-globin (ß-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and ß-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 ß-hemoglobinopathies.