Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin organization

Abstract The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human β-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the β-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

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Modulation of Chromatin Boundary Activities by Nucleosome-Remodeling Activities in Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.00183-09 ◽

2009 ◽

Vol 30 (4) ◽

pp. 1067-1076 ◽

Cited By ~ 19

Author(s):

Mo Li ◽

Vladimir E. Belozerov ◽

Haini N. Cai

Keyword(s):

Drosophila Melanogaster ◽

Nucleosome Occupancy ◽

Boundary Function ◽

Higher Order ◽

Chromatin Organization ◽

Nurd Complex ◽

Nucleosome Remodeling ◽

Chromatin Boundary ◽

Enhancer Blocking

ABSTRACT Chromatin boundaries facilitate independent gene regulation by insulating genes from the effects of enhancers or organized chromatin. However, the mechanisms of boundary action are not well understood. To investigate whether boundary function depends on a higher order of chromatin organization, we examined the function of several Drosophila melanogaster insulators in cells with reduced chromatin-remodeling activities. We found that knockdown of NURF301 and ISWI, key components of the nucleosome-remodeling factor (NURF), synergistically disrupted the enhancer-blocking function of Fab7 and SF1 and augmented the function of Fab8. Mutations in Nurf301/Ebx and Iswi also affected the function of these boundaries in vivo. We further show that ISWI was localized on the endogenous Fab7 and Fab8 insulators and that NURF knockdown resulted in a marked increase in the nucleosome occupancy at these insulator sites. In contrast to the effect of NURF knockdown, reduction in dMi-2, the ATPase component of the Drosophila nucleosome-remodeling and deacetylation (NuRD) complex, augmented Fab7 and suppressed Fab8. Our results provide the first evidence that higher-order chromatin organization influences the enhancer-blocking activity of chromatin boundaries. In particular, the NURF and NuRD nucleosome-remodeling complexes may regulate Hox expression by modulating the function of boundaries in these complexes. The unique responses by different classes of boundaries to changes in the chromatin environment may be indicative of their distinct mechanisms of action, which may influence their placement in the genome and selection during evolution.

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Differences in BCL11A SUMOylation Are Associated with Differences in γ-Globin Expression in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.458.458 ◽

2009 ◽

Vol 114 (22) ◽

pp. 458-458

Author(s):

Tatiana Kouznetsova ◽

Kestis Vaitkus ◽

Vinzon Ibanez ◽

Joseph DeSimone ◽

Donald Lavelle

Keyword(s):

Dna Methylation ◽

Western Blot ◽

Progenitor Cells ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Cells ◽

Developmentally Regulated ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Globin Promoter

Abstract Abstract 458 Increased fetal hemoglobin (HbF) levels associated with acute erythropoietic stress in man and experimental baboons have been proposed to result from increased commitment of early progenitors that preferentially express γ-globin to the terminal erythroid differentiation pathway. The increased propensity of early progenitors to preferentially express γ-globin has been hypothesized to be due to the presence of trans-acting factors favoring γ-globin expression. Because increased HbF in response to acute erythropoietic stress does not occur in transgenic human β-globin gene locus mouse models, investigation of the mechanism responsible for this phenomenon requires the use of a primate model system. We investigated the role of DNA methylation and the trans-acting factor BCL11A in the mechanism responsible for increased HbF in a primary cell culture system designed to mimic conditions associated with acute erythropoietic stress. Erythroid progenitor cells (EPC) derived from CD34+ baboon bone marrow (BM) cells cultured in Iscove's medium containing 30% fetal bovine serum supplemented with 2 U/ml Epo, 200ng/ml SCF, and 1uM dexamethasone express high levels of γ-globin (0.47+ 0.09 γ/γ+β; n=6). Bisulfite sequence analysis performed to determine whether changes in DNA methylation of 5 CpG residues within the 5' γ-globin promoter regions were associated with increased γ-globin expression showed that DNA methylation levels were similar in BM erythroid cells from normal baboons expressing very low levels of HbF (<1%), bled baboons expressing moderately elevated levels of HbF (5-10%), and cultured erythroid progenitor cells expressing highly elevated levels of HbF (30-50%). Changes in γ-globin promoter DNA methylation were thus not associated with increased γ-globin expression in EPC cultures. Further experiments were therefore performed to investigate whether differences in BCL11A expression were associated with increased γ-globin in EPC cultures. Western blot assays performed using three different anti-BCL11A monoclonal antibodies recognizing epitopes present in the N terminus, core, and C terminus detected different BCL11A isoforms in cultured EPC and normal BM erythroid cells. The size of the predominant protein band detected in cultured EPC was 125kDa, corresponding to the reported size of the in vitro transcription/translation product encoded by the BCL11A-XL transcript (Liu et al, Mol Cancer 16:18, 2006). In contrast, the size of the predominant band observed in BM erythroid cells was 220kDa. The 220kDa isoform was not observed in cultured EPC. Higher molecular weight forms of BCL11A have been observed following co-transfection of vectors encoding BCL11A and SUMO-1 (Kuwata and Nakamura, Genes Cells 13:931, 2008). Therefore we investigated whether the post-translational modification SUMOylation was responsible for the difference in the size of the 125 and 220kDa isoforms. Immunoprecipitation experiments performed using either SUMO-1 or SUMO 2/3 antibodies followed by Western blot with anti-BCL11A antibody showed that the 220 kDa isoform, but not the 125kDa isoform, was immunoprecipitated by either anti-SUMO-1 or anti-SUMO-2/3 antibody, confirming that the 220 kDA isoform, but not the 125 kDa isoform, was SUMOylated. Western blot assays performed to investigate the relative levels of these isoforms in BM erythroid cells of normal baboons, phlebotomized baboons, and early gestational age (53d) baboon fetal liver showed that expression of the 125kDa isoform was increased in bled compared to normal unbled baboons, suggesting that the deSUMOylated BCL11A isoform was increased by erythropoietic stress. The relative levels of the 125 and 220 kDa isoforms were similar in bled BM and fetal liver, indicating that SUMOylation of BCL11A was not developmentally regulated. The absolute level of BCL11A was reduced in fetal liver erythroid cells compared to BM erythroid cells consistent with observations showing that the level of BCL11A expression is developmentally regulated in man (Sankaran et al, Nature epub 2009). We conclude that BCL11A is post-translationally modified by SUMOylation in primary BM erythroid cells, but not in cultured EPC expressing high levels of HbF and suggest that modulation of the level of BCL11A SUMOylation is important in the mechanism responsible for increased HbF levels during recovery from acute erythropoietic stress. Disclosures: No relevant conflicts of interest to declare.

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Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice

Blood ◽

10.1182/blood.v97.6.1861 ◽

2001 ◽

Vol 97 (6) ◽

pp. 1861-1868 ◽

Cited By ~ 66

Author(s):

Elise Coghill ◽

Sarah Eccleston ◽

Vanessa Fox ◽

Loretta Cerruti ◽

Clark Brown ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Target Genes ◽

Fetal Liver ◽

Globin Gene ◽

Single Copy ◽

Erythroid Cell ◽

Functional Analyses ◽

Definitive Erythropoiesis ◽

Globin Gene Expression

Erythroid Kruppel-like factor (EKLF) is a transcription factor of the C2H2 zinc-finger class that is essential for definitive erythropoiesis. We generated immortal erythroid cell lines from EKLF−/− fetal liver progenitor cells that harbor a single copy of the entire human β-globin locus and then reintroduced EKLF as a tamoxifen-inducible, EKLF–mutant estrogen receptor (EKLF-ER™) fusion protein. Addition of tamoxifen resulted in enhanced differentiation and hemoglobinization, coupled with reduced proliferation. Human β-globin gene expression increased significantly, whereas γ-globin transcripts remained elevated at levels close to endogenous mouse α-globin transcript levels. We conclude that EKLF plays a role in regulation of the cell cycle and hemoglobinization in addition to its role in β-globin gene expression. The cell lines we used will facilitate structural and functional analyses of EKLF in these processes and provide useful tools for the elucidation of nonglobin EKLF target genes.

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Multiple Defects in Erythroid Gene Expression in Erythroid Krupple-Like Factor (EKLF) Target Genes in EKLF-Deficient Mice.

Blood ◽

10.1182/blood.v104.11.1602.1602 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1602-1602

Author(s):

Patrick G. Gallagher ◽

Andre M. Pilon ◽

Murat O. Arcasoy ◽

David M. Bodine

Keyword(s):

Gene Expression ◽

Pyruvate Kinase ◽

Target Genes ◽

Fetal Liver ◽

Globin Gene ◽

Subtractive Hybridization ◽

Liver Cells ◽

Fetal Liver Cells ◽

Microarray Analyses ◽

Deficient Mice

Abstract EKLF is the founding member of the KLF family of transcription factors. EKLF-deficient (−/−) mice die at d14-15 of gestation of severe anemia, attributed to decreased expression of the β-globin gene. Morphology of fetal-liver derived circulating erythroid cells in EKLF −/− mice does not mimic that seen in thalassemia, and mice expressing high levels of γ-globin bred onto an EKLF −/− background do not correct the anemia or rescue the −/− mice. These data suggest additional red cell defects are present in EKLF −/− mice contributing to the anemia. To address this hypothesis, we performed subtractive hybridization and microarray analyses with RNA isolated from d13.5 fetal livers of wild type (WT,+/+) and EKLF-deficient mice. In subtractive hybridization, WT (1X) was the tester population and EKLF −/− (30X) was the driver. >200 differentially expressed clones were sequenced. 122 clones were β-globin; 21 alpha hemoglobin stabilizing protein (AHSP); and the remainder were RBC membrane proteins including band 3 and β-spectrin. Microarray analyses were performed with Affymetrix GeneChip Mouse Genome 430 2.0 arrays; 3 independent EKLF +/+ and −/− RNA samples were analyzed. AHSP had the most significantly decreased expression in −/− samples, reduced to 5% of WT (p<0.0001). Other significantly down regulated genes, in addition to those identified by subtractive hybridization, included pyruvate kinase and ankyrin. Reduced expression in EKLF −/− RNA was confirmed using ribonuclease protection assay and/or real-time RT-PCR. AHSP mRNA was decreased by 75–90%; band 3 and β-spectrin were decreased by ~40%; ankyrin by 45%, and pyruvate kinase 15%. Flow cytometry of +/+ and −/ − fetal liver cells using TER119 revealed a TER119Hi population of cells absent in −/ − fetal liver cells, suggesting a block in differentiation to more mature erythroid progenitors. To ensure that the potential EKLF target genes were expressed in the TER119Lo population and were not identified because they were present only in the TER119Hi, the expression of each selected gene was analyzed in TER119Lo and TER119Hi cells by real time PCR. Target gene/α-globin ratios indicated the selected genes were expressed at levels >2 fold higher in TER119Lo than TER119Hi cells. Studies of the AHSP locus identified a strong DNaseI hypersensitive site (HS) in WT fetal liver nuclei between 5′ of the AHSP promoter, that was absent in −/ − chromatin. Chromatin immunoprecipitation analysis of WT fetal liver chromatin spanning 3.5kb of the AHSP locus using 13 primer pairs (~300bp intervals from >1kb 5′ and 3′) demonstrated 2 regions of hyperacetylation of histones H3 and H4. The 5′ region corresponded to the DNaseI HS, and the other region maps 3′ of the AHSP polyA signal. Histones H3 and H4 were also acetylated in the interval between these hyperacetylated regions, while the chromatin upstream and downstream of these regions were hypoacetylated. In chromatin from −/ − fetal liver cells, all sites were hypoacetylated, correlating with the severe reduction in AHSP gene expression. These results support the hypothesis that the anemia in EKLF −/ − mice is due to both decreased expression of the β-globin gene and other erythroid genes including those involved in membrane integrity, stabilization of α-globin protein, and glycolysis leading to defects in erythrocyte structure, function, and metabolism. They also suggest that EKLF may act as a transcription factor and a chromatin modulator for genes other than β-globin.

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Reactivation of Silenced Human HbF In Adult Mice by Inactivation of BCL11A

Blood ◽

10.1182/blood.v116.21.643.643 ◽

2010 ◽

Vol 116 (21) ◽

pp. 643-643

Author(s):

Jian Xu ◽

Vijay G. Sankaran ◽

Erica B. Esrick ◽

Benjamin L. Ebert ◽

Stuart H. Orkin

Keyword(s):

Dna Methylation ◽

Knockout Mice ◽

Fetal Liver ◽

Globin Gene ◽

Conditional Knockout ◽

Late Time ◽

Globin Genes ◽

Hemoglobin Switching ◽

Erythroid Precursors

Abstract Abstract 643 Persistence of fetal hemoglobin (HbF) in adults ameliorates severity of sickle cell disease and β-thalassemia. The transcriptional repressor BCL11A is a newly identified critical mediator of hemoglobin switching and HbF silencing. Previously we showed that BCL11A knockout mice with a human β-globin gene cluster transgene (β-locus mice) fail to silence mouse embryonic globins and human fetal (γ-) globins in adult erythroid cells of the fetal liver. The ratio of human fetal to adult globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that γ constitutes >90% of the β-like human expression at embryonic day (E)14.5 and >75% at E18.5. These findings provide compelling evidence that BCL11A controls hemoglobin switching in vivo. These BCL11A-null mice are postnatally lethal. Thus, the extent to which developmental silencing of HbF expression is dependent on BCL11A in adult animals cannot be assessed. Here we examined by formal genetics the contribution of BCL11A to HbF silencing through conditional inactivation of BCL11A in β-locus mice. Mice harboring erythroid-specific inactivation of BCL11A develop normally. As in the conventional knockout, the hemoglobin switching fails to occur in the fetal liver, such that γ constitutes >80% of the β-like human globins. After birth, the level of γ-globin is maintained persistently and contributes 43% in newborns, 25% in 4-week-old young adults, and 12% in 30-week-old adults. Even at this late time, the level of γ-globin is >500-fold that of control mice. The viability of these mice, taken together with ostensibly normal red cell production, indicates that BCL11A has few, if any, non-critical globin targets. To determine if loss of BCL11A in the adult reactivates γ-globin genes that were previously silenced developmentally, we conditionally inactivated BCL11A through induction of Mx1-Cre. Acute loss of BCL11A in adult bone marrows leads to persistent reactivation of γ-globin (>500-fold derepression compared to controls). Thus, BCL11A is required in vivo to maintain HbF silencing in adults. Gradual silencing of γ-globin in BCL11A-null adults suggests the presence of additional silencing pathways in the mouse trans-acting environment. In support of this hypothesis, we observed that the levels of DNA methylation at the γ-globin promoters are substantially decreased in BCL11A-null erythroid precursors from E14.5 fetal livers (40%), bone marrows of young (59%) and old (66%) mice. The levels are >80% in control mice at all ages. Loss of DNA methylation at γ-promoters indicates that developmental silencing of HbF is impaired upon loss of BCL11A. The gradual increase of DNA methylation indicates that the γ-globin genes are subject to epigenetic silencing in the absence of BCL11A in the mouse trans-acting environment. Histone deacetylases (HDACs) are potential molecular targets mediating HbF induction. By high-resolution ChIP-chip analysis, we demonstrate that HDAC1 occupies the γ-globin genes in primary human adult erythroid precursors. Administration of a HDAC inhibitor (Vorinostat) to BCL11A conditional knockout mice leads to further elevation of HbF, suggesting that the combination of BCL11A downregulation and HDAC inhibition may provide a strategy for efficient HbF augmentation. Collectively, these findings provide important insight into the role of BCL11A in HbF silencing in adults and new clues for target-based therapeutics in patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

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A global role for EKLF in definitive and primitive erythropoiesis

Blood ◽

10.1182/blood-2005-07-2888 ◽

2006 ◽

Vol 107 (8) ◽

pp. 3359-3370 ◽

Cited By ~ 140

Author(s):

Denise Hodge ◽

Elise Coghill ◽

Janelle Keys ◽

Tina Maguire ◽

Belinda Hartmann ◽

...

Keyword(s):

Target Genes ◽

Fetal Liver ◽

Globin Gene ◽

Protein Band ◽

Cytoskeletal Proteins ◽

Erythroid Cell ◽

Blood Group Antigens ◽

Red Cell Membrane ◽

Fusion Construct

Abstract Erythroid Kruppel-like factor (EKLF, KLF1) plays an important role in definitive erythropoiesis and β-globin gene regulation but failure to rectify lethal fetal anemia upon correction of globin chain imbalance suggested additional critical EKLF target genes. We employed expression profiling of EKLF-null fetal liver and EKLF-null erythroid cell lines containing an inducible EKLF-estrogen receptor (EKLF-ER) fusion construct to search for such targets. An overlapping list of EKLF-regulated genes from the 2 systems included α-hemoglobin stabilizing protein (AHSP), cytoskeletal proteins, hemesynthesis enzymes, transcription factors, and blood group antigens. One EKLF target gene, dematin, which encodes an erythrocyte cytoskeletal protein (band 4.9), contains several phylogenetically conserved consensus CACC motifs predicted to bind EKLF. Chromatin immunoprecipitation demonstrated in vivo EKLF occupancy at these sites and promoter reporter assays showed that EKLF activates gene transcription through these DNA elements. Furthermore, investigation of EKLF target genes in the yolk sac led to the discovery of unexpected additional defects in the embryonic red cell membrane and cytoskeleton. In short, EKLF regulates global erythroid gene expression that is critical for the development of primitive and definitive red cells.

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ZNF802 (JAZF1), a Possible New Therapeutic Target for Treatment of β-Thalassemia

Blood ◽

10.1182/blood-2019-127678 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3546-3546

Author(s):

Chokdee Wongborisuth ◽

Amornrat Tangprasittipap ◽

Usanarat Anurathapan ◽

Nuankanya Sathirapongsasuti ◽

Orapan Sripichai ◽

...

Keyword(s):

Therapeutic Target ◽

Molecular Mechanisms ◽

Fetal Liver ◽

Globin Gene ◽

Transcriptional Repressor ◽

P Value ◽

Gene Promoters ◽

Erythroid Cells ◽

Hemoglobin Switching ◽

Adult Hemoglobin

β-Thalassemias are groups of inherited blood disorders caused by reduced or absent production of β-globin chains of adult hemoglobin (HbA; α2β2). The severely affected patients with β-thalassemia require lifelong blood transfusion and chelation therapy. Reactivation of fetal hemoglobin (HbF; α2γ2) could ameliorate the symptoms of patients via increased total hemoglobin production and reduced imbalanced α- to non-α-globin synthesis. Hemoglobin switching during human development requires activities of several transcription factor proteins, including those bind to the globin gene promoters. However, the regulation of fetal to adult hemoglobin switching is not fully understood. Previously, we have carried out the microarray analysis comparing human erythroblasts derived from therapeutic aborted fetal liver and adult peripheral blood. The results showed that ZNF802 mRNA is among the transcripts that up-regulated in adult erythroblast of greater than 2-fold change. ZNF802, also called JAZF1, is a 27 kDa protein containing two zinc finger motifs that acting as transcriptional repressor by binding to the direct repeat (DR) elements on DNA. In this study, quantitative RT-PCR was performed in order to validate the microarray data. We observed 13-fold up-regulation of ZNF802 in adult erythroid cells compared to those cells derived from fetal liver. To further elucidate the role of ZNF802 in reduction of HbF expression in adult erythroids, the lentivirus carrying ZNF802 specific shRNA and non-targeted shRNA were designed and constructed for transduction in erythroblasts. Hematopoietic CD34+ progenitor cells were isolated from β-thalassemia/HbE patients (n=9) and subjected to ex vivo two-phase liquid erythroid culture. The results showed the reduction of greater than 90% of ZNF802 transcript and nearly undetectable level of ZNF802 protein in cells transduced with the ZNF802 shRNA lentivirus. As expected, the level of γ-globin transcript and levels of HbF were increased in ZNF802 knockdown erythroid cells in β-thalassemia/HbE (5-fold changed and HbF 26.9±7.4% vs 18.5±6.8% in non-targeted shRNA (p-value = 0.04), respectively). Interestingly, we found up-regulation of the embryonic ε-globin and ζ-globin transcripts in knockdown ZNF802 cells compared to non-targeted shRNA control (15-fold and 10-fold changed (p-value = 0.01), respectively). To note, the diminishing of ZNF802 effect on the proliferation and differentiation pattern of erythriod cells was not found. Altogether, our findings suggested that ZNF802 may play a role as the transcriptional repressor of γ-globin gene in human adult erythroid cells, thus served as a possible new therapeutic target for treatment of β-thalassemia. The molecular mechanisms underlying the ZNF802 mediated HbF reduction are underway. Disclosures No relevant conflicts of interest to declare.

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Different Hemoglobin Switching Pattern of β-Thalassemia Mutations at the Proximal and Distal Human β-Globin CACCC Box.

Blood ◽

10.1182/blood.v110.11.1780.1780 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1780-1780

Author(s):

Maria F. Marongiu ◽

Susanna Porcu ◽

Daniela Poddie ◽

Dubravka Drabeck ◽

Tom DeWit ◽

...

Keyword(s):

Fetal Development ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Yolk Sac ◽

Control Line ◽

Hemoglobin Switching ◽

Switching Pattern

Abstract The CACCC box is duplicated in the β globin gene promoter of humans and other mammals. While the function of the proximal element as a binding site for EKLF has already been well established, the role of the distal element remains unclear The distal CACCC box has been previously reported not to bind EKLF in vitro. A minor role of the distal CACCC element in β globin gene promoter function is suggested by the observation that naturally occurring β thalassemia mutations affecting the proximal CACCC box are far more severe than those affecting the distal element. Nevertheless recent evidences demonstrate: that EKLF does indeed bind to the distal CACCC motif, although with low affinity. that the CCTCACCC is required for maximal stimulation of the β-globin gene by EKLF and that silent β-thalassemia due to mutations of the distal CACCC box affects the binding and responsiveness to EKLF of the β-globin gene promoter. Our interest in the function of the distal CACCC element springs from the observation that β thalassemia mutation affecting the distal box show an age related pattern of expression being more severe in the childhood than in the adulthood. In order to get light inside the role of this element in the function of the β globin gene and in the γ to β hemoglobin switching we have analyzed the effect of mutations at the proximal and distal element “in vivo”. We have engineered, by site specific mutagenesis, the β-101 (distal CACCC element) and β-87 (proximal CACCC element) mutations inside the “minilocus “ γ-β construct. The minilocus construct has been widely used to study hemoglobin switching in vivo. This construct contains the full β-globin Locus Control Region (LCR), the Aγ globin gene, the β-globin gene and the 3′ hypersensitive site (HS) of the β-globin cluster. Three mice transgenic lines have been produced. The pattern of g versus β-globin switching has been analyzed during the development by S1 analysis and real time PCR. We have dissected the yolk sac at 10 days post conception (pc) to asses the embryonic stage of erythopoiesis; the fetal liver at 12, 14 and 16 days pc to asses the fetal stage or erythropoiesis when the g to b competitive switching take place; and the adult blood. Our results indicated that neither the β-101 nor the β-87 thalassemia mutations affect the competitive silencing of the b-globin gene in the yolk sac. During the fetal liver stage of erythropoiesis, were both human g and b human transgenes are expressed, the pattern of γ-β hemoglobin switching is striking different for the two different constructs. The b-87 minilocus γ-β construct shows a delayed switching patter mainly due to the low activation of the mutated β globin gene. The impairment of the expression of the β-87 globin gene became more severe during the fetal development compared to the control line. On the other hand the β-101 minilocus γ-β construct shows a γ-β hemoglobin switching pattern which is anticipated respect to the control line. In addition the effect of the β-101 mutation became less severe during the fetal development. These results highlight a possible role of the distal CACCC element in hemoglobin switching and in particular in the early stage of β-globin activation.

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Stage Selective Requirement For The N-Terminus Of GATA1 During Erythropoiesis

Blood ◽

10.1182/blood.v122.21.3670.3670 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3670-3670

Author(s):

Monika J Stankiewicz ◽

Timothy M Chlon ◽

John D Crispino

Keyword(s):

Down Syndrome ◽

Target Genes ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Erythroid Differentiation ◽

Full Length ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Megakaryoblastic Leukemia ◽

Exclusive Production

Abstract The X-linked blood transcription factor GATA1 is required for the survival and maturation of erythroid cells. Loss of Gata1 causes profound anemia and related mid-gestation lethality in mouse models and human GATA1 mutations are associated with congenital dyserythropoietic anemia, congenital thrombocytopenia, porphyria, Diamond-Blackfan anemia (DBA) and acute megakaryoblastic leukemia in children with Down syndrome (DS-AMKL). In DBA, DS-AMKL, and a subset of congenital anemia, GATA1 mutations cause the exclusive production of a shorter GATA1 isoform, termed GATA1s. The GATA1s isoform retains both zinc fingers and binds DNA and cofactors accordingly, but lacks the N-terminal 83 residues of full-length GATA1. Intriguingly, exclusive production of GATA1s promotes AMKL in the context of Down syndrome (trisomy 21), while disomic individuals harboring the same type of mutations suffer from anemia and severe red cell defects. A mutation causing exclusive production of Gata1s in mice does not appear to affect adult hematopoiesis, but has been shown to cause a marked expansion of megakaryocytes in the fetal liver. The effects of the replacement of Gata1 with Gata1s during fetal liver erythropoiesis, however, remain uncharacterized. To investigate the effects of exclusive Gata1s production during fetal hematopoiesis, we performed comprehensive phenotypic and mechanistic studies using Gata1s knock-in embryos. We found significant changes in myelo-erythroid differentiation beyond the known expansion of megakaryocytes. Flow cytometric analysis revealed altered erythroid differentiation at embryonic days 12.5 and 14.5, evidenced by decreased Ter119 and increased CD71 expression, characteristic of delayed erythroid maturation. Changes in expression of myelo-erythroid progenitor commitment markers were also discovered. Specifically, we observed marked decreases in pre-CFU-E and CFU-E committed progenitors and an increase in pre-GM and pre-MegE populations. Our findings are consistent with a bias towards megakaryopoiesis at the expense of erythroid commitment caused by the expression of Gata1s in place of full-length Gata1. A shift in differentiation was also observed in the embryonic granulocyte/ macrophage lineage, with an increased generation of macrophages with fewer developing granulocytes. Mechanistically, we found that expression of erythroid -specific Gata1 target genes such as Alas2, Slc4a1 and Klf1 are markedly reduced in the Gata1 knock-in erythroid cells, indicating that Gata1s is a less effective transcriptional activator than full-length Gata1. In particular, given that Klf1 functions to promote erythroid specification downstream of the megakaryocyte-erythrocyte progenitor, our results suggest that Gata1s may promote megakaryocyte differentiation at the expense of erythroid differentiation, in part, by failing to activate Klf1. Taken together, our studies demonstrate a stage-specific requirement for full-length Gata1 during embryonic erythropoiesis. Furthermore, erythroid defects associated with exclusive production of Gata1s in humans may result from an incomplete activation of the erythroid transcriptional program. Disclosures: Crispino: Sanofi: Research Funding.

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Extensively Self-Renewing Erythroid Precursors Emerge from the Embryo but Not from the Adult

Blood ◽

10.1182/blood.v112.11.2455.2455 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2455-2455

Author(s):

Samantha England ◽

Kathleen E. McGrath ◽

James Palis

Keyword(s):

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Self Renewal ◽

Erythroid Precursor ◽

Stress Erythropoiesis ◽

Erythroid Precursors

Abstract The only cell in the hematopoietic hierarchy thought to be capable of long-term selfrenewal is the stem cell. An erythroid progenitor derived from mammalian hematopoietic tissue, fetal or adult, is capable of limited proliferation (103–106 fold expansion; Bauer, 1999; Panzenböck, 1998; von Lindern, 1999). Here we report that an erythroid precursor derived from the mouse embryo is capable not only of limited, but also of extensive proliferation (~1030 fold expansion). These cells resemble proerythroblasts and basophilic erythroblasts based on their morphology, their globin gene expression profile, and their immunophenotype. While aneuploidy is not necessary for extensive proliferation, it sporadically begins to accumulate after prolonged culture. These cells are capable of massive (>100 days) daily proliferation in vitro in the presence of Epo, SCF, IGF-1, and dexamethasone. Examination of cultures lacking each of these factors support that glucocorticoids play an important role in this expansion by uncoupling erythroid precursor proliferation from maturation. Despite prolonged in vitro culture, these cells preserve their potential to fully differentiate into enucleated red blood cells with the removal of dexamethasone. Differentiation occurs over 2–3 days and is characterized by the accumulation of adult (α, β1, and β2), but not embryonic (ζ, εy, and βH1), globins. The retention of full differentiation potential despite >1030 fold expansion indicates that this proliferation represents self-renewal. To determine the developmental origin of these extensively self-renewing erythroblasts (ESREs), we initiated in vitro cultures from staged mouse embryos as well as adult tissues. E7.5 embryos, that contain primitive but not definitive erythroid progenitors, failed to generate ESREs. In contrast, ESREs can be derived from E8.5–E10.5 yolk sac and E11.5–E14.5 fetal liver. These findings along with the globin expression pattern indicate that erythroblast self-renewal is associated with definitive, but not primitive, erythropoiesis. Surprisingly, marrow from adult steady-state hematopoiesis failed to yield ESREs. Furthermore, despite the characteristics shared by stress erythropoiesis (adult spleen) and fetal erythropoiesis (liver), stress erythropoiesis only yielded erythroblasts with limited, and not extensive, self-renewal capacity. This result suggests that extensive self-renewal potential is linked either to the transient yolk sac-derived definitive erythroid lineage or to the fetal hematopoietic microenvironment. We are currently investigating the mechanisms responsible for the extensive self-renewal capacity of such lineage-restricted and mature hematopoietic precursors. Our findings raise the possibility that the expansive cellular output of the erythron within the midgestation mammalian embryo may be regulated, in part, at the level of late stage erythroid precursors.

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