Adult Erythropoiesis Research Articles

A Transgenic Mouse Model for Congenital Dyserythropoietic Anemia Type I

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.

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.

Ablation of Gata1 in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis

AbstractThe transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre–mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre–mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.

Erythroid defects in TRα−/− mice

AbstractThyroid hormone and its cognate receptor (TR) have been implicated in the production of red blood cells. Here, we show mice deficient for TRα have compromised fetal and adult erythropoiesis. Erythroid progenitor numbers were significantly reduced in TRα−/− fetal livers, and transit through the final stages of maturation was impeded. In addition, immortalized TRα−/− erythroblasts displayed increased apoptosis and reduced capacity for proliferation and differentiation. Adult TRα−/− mice had lower hematocrit levels, elevated glucocorticoid levels, and an altered stress erythropoiesis response to hemolytic anemia. Most TRα−/− animals contained markedly altered progenitor numbers in their spleens. Strikingly, 20% of TRα−/− mice failed to elicit a stress erythropoiesis response and recovered very poorly from hemolytic anemia. We conclude that an underlying erythroid defect exists in TRα−/− mice, demon-strating the importance of TRα to the erythroid compartment.

Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins

AbstractPolycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)–containing proteins, which tag hypoxia-inducible factor (HIF) α subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-α stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1−/− or Phd3−/− mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2α, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1α accumulated in both the kidney and liver. Elevated HIF-1α levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2α/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway.

Mechanism for γ-Globin Induction by SCF Is Via PKCα/ERK1/2/Nrf2 Dependent Signal Pathway To Regulate Down-Stream Transcriptional Regulators NF-Y and COUP-TFII.

Increased fetal hemoglobin has been identified to be associated with stress erythropoiesis. However, the mechanisms underlying γ-globin induction during the rapid expansion of erythroid progenitor cells have not been fully elucidated. Here we examined how intracellular signaling pathway modification of specific transcriptional regulators induced γ-globin expression in vitro cultured erythroid progenitor cells in the presence of erythropoietin and stem cell factor (SCF). We find that γ-globin induced by SCF is through a PKCα-dependent Ras/Raf/Erk1/2 signaling pathway involving activation of the transcription factor NF-Ya and inhibition of the repressor Coup-TFII. Specific inhibition of PKCα with Go6976 blocked both activation of Erk1/2 and p38 MAPK induced by SCF, and abrogated the SCF increased γ-globin gene expression. Activation of Erk1/2 plays a critical role in SCF modulated down-stream transcriptional regulators, involving regulation of γ-globin gene induction. SCF induced nuclear translocation of NF-Ya is required to activate Erk1/2 increased phosphorylation of endogenous Nrf2, which involves up-regulation of thioredoxin, and down-regulation of Coup-TFII. Inhibition of either PKCα or Erk1/2 prevented SCF induced recruitment of NF-Ya, RNA polymerase II and displacement of Coup-TFII repressor from γ-globin-promoter, indicating that the PKCα-Erk1/2 MAPK pathway contributes to SCF induced the γ-globin gene induction in adult erythropoiesis. Furthermore, consistent with this concept, SCF induced the γ-globin gene induction attenuated by inhibition of PKCα or Erk1/2 MAPK. Our data suggest that SCF stimulates the PKCα-Erk1/2 MAPK signaling pathway which regulate the downstream transcriptional activator NF-Ya and repressor Coup-TFII resulting in γ-globin reactivation in adult erythropoiesis. These observations provide the molecular pathways that take part in γ-globin augmentation during “stress erythropoiesis”.

Silencing of γ-Globin Gene Expression during Adult Definitive Erythropoiesis Is Mediated by a GATA-1 Repressor Complex.

Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between -730 to -378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A marked copy of the Aγ-globin gene (Aγm-globin) was inserted between LCR 5′ HS1 and the ε-globin gene in a human β-globin locus yeast artificial chromosome (Aγm 5′ ε β-YAC). The Aγm-globin gene was autonomously silenced in Aγm 5′ ε β-YAC transgenic mice, even in the absence of an adult β-globin gene. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC to produce a Δ1s Aγm 5′ ε β-YAC. Transgenic lines containing intact β-globin loci expressed the Δ1s Aγm-globin gene in embryonic yolk sac, fetal liver, and adult blood. To further delineate the function of the Δ1s fragment, transient transfection assays and protein-DNA interaction assays were performed. The Δ1s fragment was found to act as a repressor of a constitutively active SV40 promoter in K562 cells. DNaseI footprinting analysis and electromobility shift assays demonstrated GATA-1-binding at a site -570 bp upstream of the Aγ-globin CAP site. Recently generated β-YAC transgenic mice containing a T>G point mutation at the -570 GATA site of the normally-located Aγ-globin gene displayed a HPFH phenotype. Together, these data suggested that the -730 to -378 Aγ-globin gene region contains a silencer element at the -570 GATA site that binds a GATA-1 repressor complex during the adult stage of definitive erythropoiesis to silence expression of the Aγ-globin gene. Previous studies suggested that when GATA-1 functions as a repressor, it interacts with components of the MeCp1/NuRD complex. This complex may remodel chromatin into a repressed state, leading to silenced Aγ-globin gene expression during adult definitive erythropoiesis. The presence of components of the MeCP1/NuRD complex was assessed in uninduced (γ-globin repressor present) and induced (γ-globin repressor absent) erythroid cells (K562 and KU812) and non-erythroid cells (HFF) by Western blot analysis using an antibody to Mi2, which is a component of the NuRD complex. Mi2 protein was observed in erythroid cells when the levels of γ-globin were low (uninduced K562 or KU812 cells), whereas only a weak signal was detected when γ-globin expression was induced in these cells. The Mi2 signal in the HFF cells was even weaker. Chromatin immunoprecipitation (ChIP) using fetal liver samples from day E12 and E18 conceptuses of wild-type β-YAC transgenics showed that GATA-1, FOG-1 and Mi2 proteins co-localize to the -570 GATA site of the Aγ-globin gene in samples where γ-globin is silenced (E18 fetal liver), but not in samples where γ-globin is expressed (E12 fetal liver). Our data strongly suggest that the MeCP1/NuRD complex interacts with GATA-1 protein to form a repressor that may be involved in silencing Aγ-globin gene expression. In addition, we show that GATA-1, FOG-1 and Mi2 are recruited to the analogous -567 GATA site of Gγ-globin, in a pattern that parallels that of Aγ-globin. However the binding of these proteins to Gγ-globin is weaker than that observed for Aγ-globin. These data suggest that GATA-1-mediated repression is common to both γ-globin genes, but that other mechanisms function in the differential regulation of the two γ-globin genes.

Neither DNA Hypomethylation or Changes in the Kinetics of Erythroid Differentiation Account for 5-Azacytidine's Ability To Induce Human Fetal Hemoglobin.

5-Azacytidine (5-Aza) is among the most potent inducers of fetal hemoglobin (HbF) in patients with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first model is based on the drug's ability to inhibit DNA methyltransferase enzymes causing global DNA hypomethylation, including the promoters of the fetal globin genes, resulting in their expression during adult erythropoiesis. The second model is based on the drug's other well-know property - cytotoxicity. In this model, a cohort of differentiating erythroblasts either dies or undergoes growth arrest during drug exposure. The marrow then responds with a rapid proliferation of erythroblasts which express high levels of fetal globin mRNA and HbF. To determine which model best explains HbF induction by 5-Aza, we used an in vitro differentiation system in which CD34+ cells from normal donors are cultured with SCF, IL-3 and Flt-3 ligand for 7 days and then switched to Epo for an additional 14 days. This results in >90% erythroid cells, a 2,000-fold expansion in cell number and Hb expression similar to that seen in normal red cells (∼99% HbA, ∼1% HbF). To model common 5-Aza dosing schedules, we treated cultures daily with doses ranging from 0 to 1000 nM. Near-maximal induction of γ-globin mRNA (∼2-fold increase in γ/γ+β) and HbF (from 1% to 40%) occurred at the 300 nM dose level. While this dose was associated with hypomethylation of the 7 CpGs between −256 and +50 bp of the γ promoter (59% +/− 14% methylated CpGs vs. 92% +/− 8.1% in untreated controls, p < 0.001), no changes were found in cell expansion rate, differentiation kinetics (as judged by glycophorin A expression) or cell cycle distribution. These results argue against the cytotoxicity model. We also observed that global DNA methylation was not significantly changed by the 300 nM dose of 5-Aza. Because 5-Aza is incorporated into both RNA and DNA, it affects many cellular processes beyond DNA methylation. To determine if DNA hypomethylation is sufficient for γ-globin mRNA and HbF induction, we treated differentiating cells with DNMT1 siRNA. This resulted in transiently decreased DNMT1 mRNA and protein and lowered global DNA methylation. γ-globin promoter methylation decreased to levels equivalent to those seen with 300 nM 5-Aza (61% +/− 7% of CpGs methylated vs. 88% +/− 8% in controls, p < 0.001) but did not induce γ-globin mRNA or HbF. To further evaluate this unexpected finding we used a lentiviral vector expressing DNMT1 shRNA. This suppressed DNMT1 mRNA and protein throughout differentiation and decreased total γ-globin promoter methylation from 98% +/− 4% in control cells, to 20% +/− 11% (p<0.001) in treated cells. This level of γ promoter methylation is similar to that seen in fetal erythroid cells (13% +/− 7%). Global CpG methylation was also decreased vs. controls (30% +/− 10% vs. 83% +/− 11%, p<0.001). Again, despite significant γ promoter and global hypomethylation, no induction of γ-globin mRNA or HbF was observed. These results suggest that neither model explains 5-Aza's ability to induce fetal Hb and that alternative mechanisms await discovery. If verified, these findings have important implications for the future development of clinically useful HbF inducing agents.

Erythropoietin Lacking Hypoxia Responsive Element in 3′ Untranslated Region Stimulates Hepatic Erythropoiesis in Anemic Xenopus.

Recent studies on EPO-EPOR systems in non-mammalian vertebrate including frog and teleost fishes demonstrate extramedullary adult erythropoiesis instead of bone marrow. In adult Xenopus laevis (African clawed frog), an animal model of hepatic erythropoiesis should give an opportunity for understanding the microenvironment of vascular niche. Therefore, we investigate on erythrocyte development, gene expressions and gene regulations of EPO and EPOR molecules in adult Xenopus liver. In situ hybridization and immunostaining revealed that Xenopus EPOR (xlEPOR) expressing erythrocyte progenitors, which have low hemoglobin content, were localized among liver sinusoids. The maximum number of xlEPOR expressing cells was observed when peripheral RBC count reached a nadir after hemolytic anemia. Flowcytometric analysis of peripheral blood cells and dispersed liver cells using anti-xlEPOR antibodies also indicate that the xlEPOR+ cells in liver were increased as decreasing count of peripheral RBC. After a nadir of RBC count, xlEPOR+ immature nucleated erythrocytes were emerged in the circulation. The count of xlEPOR+ immature erythrocytes was gradually decreased as increasing count of xlEPOR− mature erythrocytes. Since mature erythrocytes are still nucleated in Xenopus, we used xlEPOR molecules as erythroid differentiation marker. Meanwhile real time RT-PCR analysis of Xenopus EPO (xlEPO) mRNA revealed that xlEpo gene expression was significantly induced in anemic liver compared to normal liver. These data suggest that xlEPO-xlEPOR signaling between erythrocyte progenitors and liver cells progress the proliferation and differentiation of erythrocyte progenitors, and the mobilization of immature erythrocytes into the circulation. Our previous study showed that the anemic serum of phenylhydrazine administrated Xenopus contains erythroid colony forming activity; however, there is no information about the relationship between anemia and hypoxia enough to stimulate erythropoiesis in adult Xenopus. In mammalian species, Epo gene expression is upregulated by binding of hypoxia inducible factor-1a (HIF-1a) and ARNT complex to hypoxia response element (HRE) located in 3′ enhancer region of Epo gene. In frog and fishes, EPO mRNAs are expressed even in normoxia condition. In fish Epo genes, consensus HRE sequence (ACGTG) were not found in 3′UTR, as well as the reporter assay failed to show Epo upregulation respond to hypoxia. Since any consensus HRE sequence was not found in 3′ UTR of xlEpo gene, we examined whether HIF-1a mediates xlEpo gene regulation. By western blot analysis of HIF-1a, we assessed whether HIF-1a is stabilized in anemia; meanwhile binding capacity of HIF-1a to 5′, 3′ UTRs and intron regions of xlEpo gene was analyzed by gel shift mobility assay. The findings in non-mammalian animal model demonstrate the basis of erythropoietic gene regulations, as well as molecular mechanism underlying adult extramedullary erythropoiesis.

Thalidomide induces γ-globin gene expression through increased reactive oxygen species–mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis

AbstractAlthough thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide’s specific effects on γ-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on γ-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34+ cells. We found that thalidomide induced γ-globin mRNA expression in a dose-dependent manner, but had no effect on β-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogen-activated protein kinase (MAPK) signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. Pretreatment of cells with the antioxidant enzyme catalase and the intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide-induced p38 MAPK activation and histone H4 acetylation. Moreover, pretreatment with catalase and DMTU diminished thalidomide-induced γ-globin gene expression. These data indicate that thalidomide induces increased expression of the γ-globin gene via ROS-dependent activation of the p38 MAPK signaling pathway and histone H4 acetylation.

Normal erythropoiesis but severe polyposis and bleeding anemia in Smad4-deficient mice

AbstractThe tumor suppressor Smad4 mediates signaling by the transforming growth factor beta (TGF-β) superfamily of ligands. Previous studies showed that several TGF-β family members exert important functions in hematopoiesis. Here, we studied the role of Smad4 in adult murine hematopoiesis using the inducible Mx-Cre/loxP system. Mice with homozygous Smad4 deletion (Smad4Δ/Δ) developed severe anemia 6 to 8 weeks after induction (mean hemoglobin level 70 g/L). The anemia was not transplantable, as wild-type mice reconstituted with Smad4Δ/Δ bone marrow cells had normal peripheral blood counts. These mice did not develop an inflammatory disease typical for mice deficient in TGF-β receptors I and II, suggesting that the suppression of inflammation by TGF-β is Smad4 independent. The same results were obtained when Smad4 alleles were deleted selectively in hematopoietic cells using the VavCre transgenic mice. In contrast, lethally irradiated Smad4Δ/Δ mice that received wild-type bone marrow cells developed anemia similar to Smad4Δ/Δ mice that did not receive a transplant. Liver iron stores were decreased and blood was present in stool, indicating that the anemia was due to blood loss. Multiple polyps in stomach and colon represent a likely source of the bleeding. We conclude that Smad4 is not required for adult erythropoiesis and that anemia is solely the consequence of blood loss.

Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation

AbstractThe mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34+ cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching.

Stem cell transplantation demonstrates that Sox6 represses εy globin expression in definitive erythropoiesis of adult mice

Sox6, a member of the Sox transcription factor family, is essential for the silencing of epsilon y globin gene expression in definitive erythropoiesis of mice. Homozygous Sox6-null mice are neonatally lethal, precluding analysis at later stages. We created adult mice that are deficient in Sox6 specifically in hematopoietic tissues by transplanting embryonic liver stem cells from Sox6-deficient mice into lethally irradiated congenic wild-type adult mice. The mice receiving mutant stem cells (mutant engrafted) showed high expression levels of epsilon y in bone marrow, spleen, and circulating blood compared with mice receiving wild-type and heterozygous stem cells (control engrafted). The level of expression of epsilon y in circulating blood was directly correlated with the percentage of successful mutant donor cell engraftment. Additionally, the mutant engrafted adult mice showed an increase in erythroid precursor cells in bone marrow, spleen, and blood. Thus, Sox6 continues to function as a major regulator of epsilon y in adult definitive erythropoiesis and is required for normal erythrocyte maturation. Therefore, Sox6 may provide a novel therapeutic target by reactivating epsilon y in patients with hemoglobinopathies such as sickle cell anemia and beta-thalassemia.

Acute postnatal ablation of Hif-2alpha results in anemia.

Adaptive transcriptional responses to oxygen deprivation (hypoxia) are mediated by the hypoxia-inducible factors (HIFs), heterodimeric transcription factors composed of two basic helix-loop-helix-PAS family proteins. The transcriptional activity of HIF is determined by the hypoxic stabilization of the HIF-alpha proteins. HIF-1alpha and HIF-2alpha exhibit high sequence homology but have different mRNA expression patterns; HIF-1alpha is expressed ubiquitously whereas HIF-2alpha expression is more restricted to certain tissues, e.g., the endothelium, lung, brain, and neural crest derivatives. Germ-line deletion of either HIF subunit is embryonic lethal with unique features suggesting important roles for both HIF-alpha isoforms. Global deletion of Hif-2alpha results in distinct phenotypes depending on the mouse strain used for the mutation, clearly demonstrating an important role for HIF-2alpha in mouse development. The function of HIF-2alpha in adult life, however, remains incompletely understood. In this study, we describe the generation of a conditional murine Hif-2alpha allele and the effect of its acute postnatal ablation. Under very stringent conditions, we ablate Hif-2alpha after birth and compare the effect of acute global deletion of Hif-2alpha and Hif-1alpha. Our results demonstrate that HIF-2alpha plays a critical role in adult erythropoiesis, with acute deletion leading to anemia. Furthermore, although HIF-1alpha was first purified and cloned based on its affinity for the human erythropoietin (EPO) 3' enhancer hypoxia response element (HRE) and regulates Epo expression during mouse embryogenesis, HIF-2alpha is the critical alpha isoform regulating Epo under physiologic and stress conditions in adults.

Protein kinase Cα is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the Aγ globin‐promoter in cellular models of hemoglobin switching

PKCalpha was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Silencing of Aγ-globin gene expression during adult definitive erythropoiesis is mediated by GATA-1 binding

Silencing of Aγ-globin gene expression during adult definitive erythropoiesis is mediated by GATA-1 binding

The transcription factor KLF11 can induce γ‐globin gene expression in the setting of in vivo adult erythropoiesis

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Thalidomide Induces gamma-Globin Gene Expression in Adult Primary Erythroid Cells through Increased Intracellular Reactive Oxygen Species Mediated Activation of p38 MAPK Signal Pathway.

Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide's therapeutic effect are still being defined. We hypothesize that thalidomide induce the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin gene expression in a dose-dependent manner. The averaged gamma/gamma+beta percentage ratio was 12.89% ± 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% ± 0.07% in Epo alone (P<0.01). Interestingly, we found that intracellular ROS level was significantly increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner. Thalidomide also increased the histone H3 phospho-acetylation and histone H4 acetylation. In contrast, treatment with the anti-oxidant N-acetylcycteine (NAC) inhibited the thalidomide induced p38 activity and histone H3, H4 acetylation. These data indicate that thalidomide induced the gamma-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.

HRI Protects Erythroid Precursors in Iron Deficiency and in β-Thalassemia by Maintaining GATA-1 and Fog-1 Expressions.

HRI is the heme-regulated eIF2a kinase that controls the translation of mRNAs in erythroid precursors by phosphorylating the a-subunit of eukaryotic translational initiation factor 2 (eIF2a). HRI is essential for translational regulation of a and b globins and the survival of erythroid precursors in iron and heme deficiency. Furthermore, HRI modifies the severity of β-thalassemic mice that are devoid of the b-globin major gene. To gain insights into the molecular mechanisms by which HRI exert its protection during stress erythropoiesis, micrroarray gene profilings of erythroid precursors were carried out under the conditions of iron deficiency and HRI deficiency. HRI +/+ (Wt) and −/ − (Ko) mice were maintained in an iron deficient diet for 2 months and during gestation to induce iron deficiency in developing embryos. Under these conditions, both Wt and Ko fetal liver cells at E14.5 exhibited impaired erythroid differentiation, increased proliferation and elevated apoptosis, hallmarks of ineffective erythropoiesis. All these parameters were aggravated by HRI deficiency. Furthermore, examinations of embryonic blood smears and fetal liver cytospins revealed the presence of inclusions in reticulocytes of Ko-Fe mice (HRI−/− in iron deficiency). These phenotypes of Ko-Fe in fetal definitive erythropoiesis were the same as those observed earlier in adult erythropoiesis. Hence, mRNAs from E14.5 fetal liver nucleated erythroid precursors were used for mouse expression arrays. In Wt-Fe, expression of 213 genes was altered more than 2-fold. The number of genes altered in Ko-Fe increased dramatically by 15-fold to 3,135. In contrast, the number of the genes changed in Ko+Fe was relatively small (85), consistent with the mild phenotype in Ko+Fe mice. Therefore, HRI is crucial in maintaining proper gene expression in iron deficient erythroid precursors. Large numbers of genes altered in Ko-Fe were involved in cell death, protein synthesis, cancer and the cell cycle, consistent with the phenotypes of elevated apoptosis and increased proliferation in Ko-Fe erythroid precursors. In addition, there were more known erythroid genes (29) and iron/heme homoeostasis genes (12) that were changed in Ko-Fe as compared to Wt-Fe. Most of these genes were targets of GATA-1. When gene profiling of Ko-Fe erythroid precursors was compared with that of G1E-Er4 cells upon induction of GATA-1 by estradiol, there were 892 overlapping genes affected in both cases. GATA-1 mRNA and protein levels in fetal liver cells were reduced in iron deficiency and in b-thalassemia, and were decreased further in HRI deficiencies. Fog-1 mRNA level was not significantly altered in iron deficiency or b-thlassemia alone, but were reduced significantly upon combined deficiencies with HRI. These results demonstrate for the first time the impairment of GATA-1 and Fog-1 expressions in iron deficiency and in b-thalassemia with deletion of the b-globin gene. Furthermore, in iron deficiency and b-thalassemia HRI is necessary to maintain both expressions of GATA-1 and Fog-1, which are essential for definitive erythropoiesis.