Elevated Levels Of γ-Globin Gene Promoter 5-Hydroxymethylcytosine are Associated With Increased DNA Hypomethylation and HbF Expression

Abstract DNA methylation of the γ-globin gene promoter represses γ-globin expression in adult-stage erythroid cells while high level γ-globin expression in fetal liver erythroid cells is associated with DNA hypomethylation. Previously we showed that DNA demethylation of the γ-globin gene promoter during fetal liver erythroid differentiation is responsible for the nearly complete loss of DNA methylation, while much more limited DNA demethylation during adult bone marrow (BM) erythroid differentiation maintains a relatively high level of γ-globin promoter DNA methylation (Singh et al Exp Hematol 35:48, 2007). As recent studies have shown the importance of 5-hydroxymethylcytosine (5-hmC) as an intermediate in active and passive mechanisms of DNA demethylation that alter epigenetic modifications regulating development and hematopoietic differentiation, experiments were performed to 1) investigate the hypothesis that DNA demethylation of the γ-globin promoter during erythroid differentiation involves 5-hmC, and 2) evaluate the role of 5-hmC in γ-globin gene expression. Levels of 5-hmC and 5-methylcytosine (5-mC) located at a CpG residue within the context of a HpaII site within the 5' γ-globin promoter were measured in 1) baboon BM cells enriched for different stages of erythroid differentiation, and 2) CD34+ BM-derived erythroid progenitors expressing high levels of γ-globin grown in liquid culture or expressing low levels of γ-globin in co-culture with the AFT024 cell line. Analysis of BM cells showed that CD117+CD36+ BM cells enriched in clonogenic late BFUe/CFUe had nearly 3 fold higher levels of γ-globin promoter 5-hmC (6.91+1.41%) compared to BM-derived erythroid precursors (2.57+0.75%; p<0.0001). In erythroid precursors expressing low levels of HbF derived from CD34+ BM cells grown in co-culture with the AFT024 murine fetal liver cell line, the levels of γ-globin promoter 5-hmC (1.72+1.19%) and 5-mC (64.84+9.22%) were not significantly different from BM-derived erythroid precursors. In contrast, the level of γ-globin promoter 5-hmC in erythroid precursors derived from CD34+ BM cells grown in liquid cultures expressing elevated levels of HbF was significantly higher (6.18+1.35%) than terminal erythroid precursors from either adult BM (p<.0001) or AFT024 co-cultures (1.72+1.19%; p<.0001) but was not significantly different than the level in CD117+CD36+ BM cells. Levels of γ-globin promoter 5-hmC were similar in erythroid precursors from liquid cultures on d7 (5.57+1.24%), d11 (6.13+1.02%), d14 (6.73+1.74%), and more primitive d7 gly- basophilic erythroblasts (6.21+1.38%). The level of DNA methylation (5-mC) was significantly less in erythroid precursors derived from liquid cultures (40.37+14.33%) compared to erythroid precursors derived from adult BM (63.10+7.72%; p<0.0005), AFT024 co-cultures (64.84+9.22%; p<0.001) and CD117+CD36+ BM cells (67.71+7.45%; p<0.002). Reduced levels of 5-mC were observed in erythroid precursors from liquid cultures on d14 (34.87+14.67%) compared to d7 (48.64+15.56%; p<0.055) suggesting that the γ-globin gene is progressively demethylated during erythroid differentiation in liquid culture. We conclude that γ-globin promoter 5-hmC levels are modulated during adult BM erythroid differentiation with 3 fold higher levels in CD117+CD36+ cells enriched in late BFUe/CFUe compared to erythroid precursors. Similar levels of γ-globin promoter 5-hmC, 5-mC, and HbF are observed in adult BM erythroid precursors and erythroid precursors derived from AFT024 co-cultures. In contrast, high levels of levels of γ-globin promoter 5-hmC, similar to levels in CD117+CD36+ BM cells, are sustained in erythroid precursors derived from liquid cultures of CD34+ BM cells and are associated with decreased γ-globin promoter 5-mC and increased HbF. Supplementation of cultures with ascorbic acid, a co-factor of the TET oxygenases that catalyze 5-hmC, reduced levels of γ-globin promoter 5-mC (20.94+9.77%) compared to controls (51.24+14.61%; p<.025) and increased γ-globin expression. These results support the hypothesis that DNA demethylation of the γ-globin promoter during erythroid differentiation, resulting in high HbF expression, occurs through a 5-hmC-mediated mechanism subject to developmental regulation by factors in the micro-environment. Disclosures: No relevant conflicts of interest to declare.

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Dynamic Regulation of 5-Hydroxymethylcytosine At the γ-Globin Promoter During Erythroid Differentiation

Blood ◽

10.1182/blood.v120.21.824.824 ◽

2012 ◽

Vol 120 (21) ◽

pp. 824-824

Author(s):

Maria Armila Ruiz ◽

Kestis Vaitkus ◽

Aparna Vasanthakumar ◽

Angela Rivers ◽

Tatiana Kouznetsova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Bone Marrow ◽

Globin Gene ◽

Erythroid Differentiation ◽

Dna Demethylation ◽

Pcr Analysis ◽

Erythroid Precursor ◽

Erythroid Precursors ◽

Globin Promoter

Abstract Abstract 824 DNA methylation is a key element responsible for γ-globin gene repression in adult erythroid cells. Our laboratory previously observed that the γ-globin gene promoter region was demethylated in a progressive manner as γ-globin expression was activated during erythroid differentiation of primary baboon pre-switch fetal liver cells and, to a lesser extent, of adult baboon bone marrow (BM) cells (Singh et al Exp Hematol 35:48-55, 2007). The mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation remains unknown. Recent studies have shown that DNA demethylation in the early embryo is mediated by the “sixth base” 5-hydroxymethylcytosine (5-hmC) whose formation from 5-methylcytosine is catalyzed by the enzymatic activity of the three TET proteins. To investigate the hypothesis that 5-hmC mediates DNA demethylation of the γ-globin promoter during erythroid differentiation, levels of 5-hmC at Msp I (CCGG) sites within the γ- and ϵ-globin promoters and γ-globin IVS II region in DNA isolated from peripheral WBC, purified terminal erythroid precursors, and FACS-purified adult bone marrow subpopulations enriched for different stages of differentiation (CD117+ CD36-, CD117+ CD36+, and CD117-CD36+), were analyzed using a T4 glucosylase-MspI quantitative real time PCR assay. Levels of 5-hmC associated with the γ-globin promoter MspI site were 11.6 fold higher (p<.0001) in terminal erythroblasts (n=9) compared to peripheral blood WBC (n=4) while 5-hmC levels associated with the ϵ-globin promoter were 11.8 fold higher (p<.0001) in terminal erythroid precursors (n=13) compared to peripheral WBC (n=4). Levels of 5-hmC associated with the γ-globin promoter (n=9) were 9.4 fold higher (p<.0001) than with the IVS II region of the γ-globin gene (n=8) in terminal erythroid precursors suggesting that elevated levels of 5-hmC within the β-globin gene complex in terminal erythroid precursors may be localized to promoter regions. Genomic 5-hmC levels, analyzed by HPLC-MS, were similar in WBC and terminal erythroid precursors. Within purified BM subpopulations enriched for different stages of erythroid differentiation, levels of 5-hmC associated with the γ-globin promoter were 3.2 to 4.1 fold higher in the CD117+CD36+ subpopulation enriched in erythroid colony forming cells (n=4) than in CD117+CD36- (n=3; p<.01), more differentiated CD117-CD36+ (n=3; p<.02), and terminal erythroid precursor (p<.001) subpopulations. Similar differences in levels of 5-hmC associated with the ϵ-globin promoter were also observed between these subpopulations. Enrichment of 5-hmC within the β-globin locus in the CD117+CD36+ and terminal erythroid precursors compared to peripheral WBC was confirmed by 5-hmC affinity selection and PCR analysis. In addition, similar differences in genomic 5-hmC levels between these subpopulations were also observed by HPLC-MS analysis. Bisulfite sequence analysis showed that the changes in 5-hmC levels at the γ-globin promoter were temporally associated with loss of DNA methylation within the γ-globin promoter as erythroid differentiation progressed. TET gene expression analysis showed that TET2 expression was 3 fold less (p<.01) while TET3 expression was >7 fold higher (p<.05) in terminal erythroid precursors compared to the CD117+CD36- subpopulation. These results strongly suggest that differences in 5-hmC associated with the ϵ- and γ-globin promoters are the result of wider dynamic alterations of genomic 5-hmC levels during erythroid differentiation that may be mediated by differences in TET gene expression and support the hypothesis that 5-hmC is involved in the mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation. Disclosures: Godley: Celgene: Research Funding.

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Developmental and Differentiation-Specific Patterns of γ- and β-Globin Promoter DNA Methylation in Primary Human Erythroid Cells.

Blood ◽

10.1182/blood.v108.11.1586.1586 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1586-1586

Author(s):

Christine Richardson ◽

Rodwell Mabaera ◽

Christopher H. Lowrey

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Globin Promoter ◽

Methylation Patterns ◽

Adult Bone

Abstract Despite years of investigation in a variety of experimental systems, the mechanisms underlying human β-globin locus developmental gene switching remain elusive. Several lines of evidence implicate DNA methylation in this process. As an initial step in studying the role of epigenetic modifications in the human switching process and in determining the mechanisms by which DNA methyltransferase inhibitors reverse the switch, we have characterized the DNA methylation patterns of the individual CpGs in the γ- and β-globin promoters in fetal liver (FL) and adult bone marrow (BM) primary erythroid cells and during in vitro differentiation of adult erythroid cells. Using the bisulfite conversion method we evaluated all CpGs in the ~500 bp regions centered on the γ- and β-globin promoter start sites. Fetal liver (FL) and adult bone marrow (BM) samples were obtained using IRB approved protocols and informed consent procedures. Erythroid cells were purified using anti-glycophorin A (glyA) magnetic beads. Purity was confirmed to be greater than 95%. Samples from five independent BM and FL samples were analyzed and 8–20 bisulfite converted sequences were determined for each promoter in each sample. Our results show that all 8 CpGs between −249 and +210 of the Gγ and Aγ-globin promoters are less than 20% methylated in FL and greater than 80% methylated in BM except for the −158 CpG which is only 40% methylated in BM(p<0.002). The 6 CpGs between −415 and +110 of the β-globin promoter show an inverse pattern with lower levels of DNA methylation in BM. Histone H3 acetylation of the γ-globin promoter, as determined by ChIP analysis, showed a complimentary pattern with higher levels in FL than BM. We next evaluated γ-globin promoter methylation patterns during in vitro erythroid differentiation from CD34+ BM cells. In this experiment, cells were grown with SCF, Flt3 ligand and IL-3 for 7 days and then in EPO for 14 days producing erythroid cells which express 99%HbA and 1% HbF. The initial day 0 CD34+ cells showed 90–100% methylation of all γ promoter sites. By day 3 in culture, before the initiation of erythroid differentiation, methylation at all sites upstream of the promoter had decreased to less than 60% and the CpG at −53 (the site of Stage Selector Protein complex binding) had decreased to less than 20%. The three CpG sites down-stream of the promoter (+6, +17 and +50) remained highly methylated. The pattern was unchanged at day 10, early in erythroid differentiation, when γ-globin mRNA expression was beginning. By day 14, when β-globin expression was peaking, methylation of the upstream promoter had increased back to the 70–100% level at all CpGs. These experiments provide a comprehensive picture of γ- and β-globin promoter methylation during the fetal and adult stages of erythroid development and of the γ-globin promoter during adult erythroid differentiation. The finding of transient γ-promoter hypomethylation during differentiation offers a potential mechanism to explain the transient γ-globin gene expression seen during normal adult erythropoiesis. Our results also raise the possibility that, just as domains of altered histone modification exist in β-globin gene loci, there may also be developmentally-specific domains of DNA methylation.

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The Tet Dioxygenase Co-Factor Ascorbic Acid Reduces DNA Methylation and Increases Expression of the γ-Globin Gene and Acts in a Combinatorial Manner with HbF-Inducing Drugs Targeting Repressive Epigenetic Modifications

Blood ◽

10.1182/blood.v124.21.334.334 ◽

2014 ◽

Vol 124 (21) ◽

pp. 334-334

Author(s):

Maria Armila Ruiz ◽

Angela Rivers ◽

Kestis Vaitkus ◽

Vinzon Ibanez ◽

Robert E. Molokie ◽

...

Keyword(s):

Dna Methylation ◽

Ascorbic Acid ◽

Dna Methyltransferase ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Differentiation ◽

Mean Difference ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Globin Promoter

Abstract Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). Differences in DNA methylation of the γ-globin gene promoter between adult and fetal liver erythroid cells are highly associated with developmental differences in γ-globin expression. Mechanisms that establish and/or modulate DNA methylation of the γ-globin promoter during adult and fetal erythroid differentiation are important in the regulation of γ-globin expression. Pharmacological manipulation of DNA methylation increases HbF in nonhuman primates and SCD patients. Decitabine, a DNA methyltransferase inhibitor that demethylates DNA and increases HbF, is currently in clinical trials. Recent studies have shown that 5-hydroxymethylcytosine (5-hmC), an oxidative product of 5-methylcytosine (5-mC) catalyzed by activity of the TET dioxygenase family, is an intermediate in developmental processes that demethylate DNA. Previously we showed that the γ-globin gene promoter was demethylated during fetal liver erythroid differentiation and to a lesser extent during adult bone marrow (BM) erythroid differentiation. We have investigated the role of 5-hmC in the mechanism of γ-globin gene demethylation by analyzing 5-hmC levels at the HpaII site located at position -51 5’ to the γ-globin transcription start site using a T4-MspI assay in DNA isolated from FACS-purified subpopulations of baboon BM cells enriched for different stages of erythroid lineage differentiation. Levels of 5-hmC were >3 fold higher (p<0.001) in the CD117+CD36+ subpopulation enriched in CFUe (7.15+1.34%) compared to the terminal erythroid precursors (2.33+0.84%) showing that 5-hmC levels are dynamically regulated during erythroid differentiation. Although baboon BM erythroid subpopulations express both TET2 and TET3, higher levels of TET3 were observed in terminal erythroid precursors than in the more primitive CD117+CD36+ subpopulation. High levels of TET3 were also observed in FACS-purified erythroid cells derived from cultured CD34+ baboon BM, human peripheral blood, and human cord blood cells suggesting a role for TET3 in erythroid differentiation. To investigate the relationship between 5-hmC, 5-mC, and γ-globin expression, levels of γ-globin promoter 5-hmC and 5-mC were determined in purified erythroid cells derived from baboon BM CD34+ erythroid progenitors grown in culture conditions resulting in either high (liquid culture) or low (AFT024 murine fetal liver stromal cell line co-culture) levels of γ-globin expression. Levels of γ-globin promoter 5-hmC (mean difference 4.93% total cytosine; p<0.005) and γ-globin chain expression (mean difference γ/γ+β=0.44; p<0.001) were higher and γ-globin promoter 5-mC levels lower (mean difference -25.2% total cytosine; p<0.01) in erythroid progenitors grown in liquid cultures compared to stromal cell line co-cultures. Supplementation of culture media with ascorbic acid, a co-factor of the TET dioxygenases, increased γ-globin expression (mean difference γ/γ+β=0.12; p<0.005) and reduced the level of γ-globin promoter DNA methylation (mean difference -29.0% total cytosine; p<0.001) in baboon BM erythroid progenitors grown in both liquid and co-cultures compared to untreated controls. Ascorbic acid also increased γ-globin expression in cultures derived from human peripheral blood CD34+ progenitors (mean difference γ/γ+β=0.08; p<0.05). In addition, in baboon BM erythroid progenitor cultures ascorbic acid increased γ-globin expression in an additive manner in combination with either the DNA methyltransferase inhibitor decitabine (p<0.001) or the LSD1 inhibitor tranylcypromine (p<0.001) compared to either drug alone, while no combinatorial effects on γ-globin expression were observed with hydroxyurea. These results demonstrate that ascorbic acid is a DNA hypomethylating agent that increases γ-globin gene expression and are consistent with a role for the TET-mediated 5-hmC pathway in the regulation of DNA methylation and expression of the γ-globin gene. Furthermore, these results suggest that vitamin C deficiency, observed in approximately 50% of patients with sickle cell disease, may limit HbF induction by drugs that target epigenetic silencing mechanisms. Disclosures No relevant conflicts of interest to declare.

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Polymorphic Pvu II Sites in the Stage-Selector Elements of the Baboon (P. anubis) γ-Globin Gene Promoter May Determine HbF Levels by Reducing DNA Methylation Density.

Blood ◽

10.1182/blood.v104.11.1211.1211 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1211-1211

Author(s):

Donald Lavelle ◽

Mahipal Singh ◽

Kestas Vaitkus ◽

Maria Hankewych ◽

Lauren Wilson ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Sequence Analysis ◽

Nucleotide Sequence ◽

Dna Binding ◽

Globin Gene ◽

Gene Promoter ◽

Methylated Dna ◽

Erythroid Precursors ◽

Globin Promoter

Abstract Phylogenetic footprinting studies previously identified conserved CpG residues in the γ-globin promoter in all simian primates that express the γ-globin gene during the fetal period of development that are absent in prosimians that express this gene during the embryonic period. (Tagle et al, J Mol Biol203:439–455, 1988). These CpG residues are methylated in adults but unmethylated in fetal tissues expressing the γ-globin gene. Reactivation of HbF synthesis in adults following treatment with the DNA methyltransferase inhibitor decitabine suggests that methylation of these CpG residues in adults may be instrumental in silencing the γ-globin gene. Sequence analysis of the baboon γ-globin gene promoter isolated from baboon (P. anubis) white blood cell DNA lambda phage libraries identified differences between the baboon and other simian primates in the nucleotide sequence at two of these CpG sites located at −51 and +16 that result in a CpG to CpA change at both positions in the baboon. At both positions these nucleotide sequence differences introduce new Pvu II sites. Three subspecies of baboons, P. anubis (n=12), P. cynocephaus (n=6) and P, hamadryas (n=6) were genotyped for the presence or absence of these Pvu II sites in the γ-globin gene promoter. Pvu II sites were detected in the γ-globin promoters of all baboons and were not present in Macaca mullatta (n=2). Individual baboons differed in the number and arrangement of polymorphic Pvu II sites. Both polymorphic sites are located in regions previously reported to act as stage selector elements (Jane et al, EMBO J11:2961–2969, 1992; Amrolia et al, J Biol Chem270:12892–12898, 1995). A family of methylated DNA binding proteins that bind methylated CpG residues and act as transcriptional repressors has been described. Repression of transcription by MeCP2, one member of this family, depends on the density of DNA methylation of the promoter region. MeCP2 loses the ability to repress transcription when the density of methyl CpG residues is reduced to <1 per 100bp (Nan et al, Cell88:471–481, 1994). Therefore we investigated the effect of these polymorphisms in combination with decitabine treatment on methylation density of the γ-globin gene promoter. Bisulfite sequence analysis of DNA obtained from erythroid precursors purified from bone marrow aspirates obtained pre- and post-treatment was performed. In pre-treatment samples obtained from two phlebotomized baboons, HbF levels were 5.4% and 9.0% and γ-globin promoter DNA methylation densities (mCpG/100bp) were 2.8 and 2.0 respectively. Following decitabine treatment (0.52mg/kg/day; 10–13d sc), HbF levels rose to 51.1% and 76% corresponding to respective DNA methylation densities of 0.85 and 0.95. High-level reactivation of the γ-globin promoter is therefore associated with a reduction in the DNA methylation density of the γ-globin promoter to <1 in purified bone marrow erythroid precursors suggesting that MeCP2 or a similar methylated DNA binding protein may repress γ-globin gene expression. We hypothesize that these polymorphisms contribute to the strong HbF response to hematopoietic stress and pharmacological agents of the baboon, compared to human, by reducing the DNA methylation density of the γ-globin gene promoter.

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

Blood ◽

10.1182/blood-2007-01-068635 ◽

2007 ◽

Vol 110 (4) ◽

pp. 1343-1352 ◽

Cited By ~ 43

Author(s):

Rodwell Mabaera ◽

Christine A. Richardson ◽

Kristin Johnson ◽

Mei Hsu ◽

Steven Fiering ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Cells ◽

Promoter Dna Methylation ◽

Globin Promoter ◽

Promoter Dna

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.

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Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6634 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6634-6640 ◽

Cited By ~ 30

Author(s):

Denise E. Sabatino ◽

Amanda P. Cline ◽

Patrick G. Gallagher ◽

Lisa J. Garrett ◽

George Stamatoyannopoulos ◽

...

Keyword(s):

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Yolk Sac ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Mrna ◽

Globin Promoter ◽

In Erythroid Cells

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

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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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The Effects of Environmental Cues on Hematopoietic Stem Cell Colony Fetal Hemoglobin Production.

Blood ◽

10.1182/blood.v116.21.3860.3860 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3860-3860

Author(s):

Jennifer L Nichols ◽

Donald Lavelle ◽

Joseph DeSimone

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Globin Gene ◽

Gene Promoter ◽

Feeder Layer ◽

Liquid Media ◽

Feeder Layers ◽

Globin Promoter ◽

Cells Cultured ◽

In Cells

Abstract Abstract 3860 Objective. Increased fetal hemoglobin (HbF) levels are associated with increased life span and reduced pain crises in patients with Sickle Cell disease. An increased understanding of the mechanisms controlling HbF expression would be important to develop new therapies to increase HbF. The objective of these experiments was to test the hypothesis that interactions between bone marrow (BM) erythroid progenitor cells and the stromal microenvironment influence HbF expression. Materials and Methods. Baboon CD34+ BM cells were harvested and purified by immunomagnetic column chromatography using the 12.8 anti-CD34 mouse monoclonal antibody and immunomagnetic microbeads conjugated to rat anti-mouse IgM (Miltenyi). CD34+ BM Cells were grown in liquid cultures in Iscove's media containing 30% fetal bovine serum, 200ng/ml stem cell factor, 2u/ml erythropoietin, and 1uM dexamethasone in the presence of an AFT024 mouse fetal liver cell line and an OP9 mouse bone marrow cell line as feeder layers, in methylcellulose media, and in liquid media. Globin chain synthesis in cultures was measured on d11 and d14 by biosynthetic radiolabeling. Cells were incubated overnight in leucine-free α-MEM media containing 50uCi/ml [3H leucine]. Globin chains were separated by High Performance Liquid Chromatography (HPLC), and the radioactivity in each fraction was determined by liquid scintillation counting. To correlate changes in γ-globin expression with DNA methylation of the γ-globin gene promoter, the methylation state of 5 CpG sites within the γ-globin promoter region was determined by bisulfite sequencing. Erythroid cells were purified from cultures on d14 by immunomagnetic column chromatography using mouse anti-baboon RBC monoclonal antibody. DNA was isolated from purified erythroid cells using Qiagen kits. Bisulfite modification was performed using Epitect bisulfite kits. Two rounds of PCR amplification were performed using nested primers flanking 5 CpG residues within the baboon γ-globin gene promoter. Minilysate DNA was prepared from at least 10 independent clones for each sample. Sequence analysis of purified DNA samples was performed at the University of Illinois DNA Sequence facility. Results. Elevated levels of γ-globin chain synthesis were observed in cells cultured in methylcellulose and liquid media in the absence of a feeder layer when compared to cells grown in the presence of feeder layers. On d11, the γ/γ+β chain synthetic ratio was 0.623 in cells cultured in methylcellulose and 0.324 in cells cultured in liquid media, compared to 0.092 in cells cultured in the presence of AFT024 feeder layers and 0.178 in cells cultured in the presence of OP9 feeder layers. On d14, the γ/γ+β chain synthetic ratio was 0.663 in cells cultured in methylcellulose and 0.349 in cells cultured in liquid media, compared to 0.135 in cells cultured in the presence of AFT024 feeder layers and 0.252 in cells cultured in the presence of OP9 feeder layers. The level of DNA methylation (%dmC) of 5 sites in the γ-globin gene promoter negatively correlated with levels of HbF production. On d14, the level of DNA methylation was 79% in cells cultured in methylcellulose, 72% in cells cultured in liquid media, and 97% in cells grown in the presence of AFT024 feeder layers. Conclusions. Cells grown in the presence of AFT024 feeder layers expressed physiologic levels of γ-globin and cells grown in the presence of OP9 feeder layers expressed midrange levels of γ-globin. Significantly increased γ-globin expression was observed in cells cultured in the absence of a feeder layer. The γ-globin promoter in cells grown in the presence of the AFT024 feeder layer exhibited a significantly higher degree of methylation than in cells grown in methylcellulose and liquid media. These results show that interactions between erythroid progenitor cells and the stromal microenvironment can influence both the level of γ-globin expression and the DNA methylation of the γ-globin gene promoter. These results have clinical relevance; if the bone marrow microenvironment could be effectively altered in vivo, methylation of the γ-globin promoter could be decreased and HbF production increased. Further experiments must be performed to determine what is mediating the methylation of the γ-globin promoter when cells are grown on these feeder layers. Disclosures: No relevant conflicts of interest to declare.

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Decitabine Targets the Erythroid Progenitor/Precursor Subpopulations for the γ-Globin Gene Demethylation in Baboon.

Blood ◽

10.1182/blood.v108.11.556.556 ◽

2006 ◽

Vol 108 (11) ◽

pp. 556-556

Author(s):

Mahipal Singh ◽

Kestas Vaitkus ◽

Donald Lavelle ◽

Maria Hankeywich ◽

Nadim Mahmud ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Differentiation ◽

Cell Subpopulation ◽

Gene Methylation ◽

Adult Bone Marrow ◽

Pre Treatment ◽

Adult Bone

Abstract The DNA demethylating drug (5–Az–2′–deoxycytidine) elevates fetal hemoglobin (HbF) to therapeutic levels in patients with sickle cell disease. To further investigate the mechanism of action of this drug and the role of DNA methylation in γ–globin gene silencing, we have analyzed the level of methylation of five CpG sites in the 5′ region of the γ–globin gene in highly purified subpopulations of cells representing different stages of erythroid differentiation from baboon (P. Anubis) using bisulfite sequencing. Baboons were treated with three different doses of decitabine (0.52, 0.26, 0.17mg/kg/day) for 10 consecutive days and pre-treatment and post-treatment adult bone marrow (ABM) were analyzed. Fetal liver (FL;n=2) and ABM cells were purified by depletion of the erythroblast subpopulation using an anti-RBC antibody (Pharmingen) in combination with immunomagnetic columns (Miltenyi) and FACS purification of CD34+CD36−, CD34+CD36+ and CD34− CD36+ subpopulations. Clonal analysis of sorted subpopulations demonstrated enrichment of CFUe in the CD34−CD36+ subpopulation, BFUe in the CD34+CD36+ subpopulation and both BFUe and CFU-GM in the CD34+CD36− subpopulation, thus confirming that these sorted subpopulations were enriched for the cells representing different stages of erythroid differentiation. A progressive decrease in the level of γ-globin gene methylation, as the degree of differentiation increased, was observed in the subpopulations purified from FL (Table 1). In pre-treatment ABM the level of γ-globin gene methylation was significantly (P<0.05) reduced in erythroblasts when compared to the CD34+CD36− subpopulation. Decitabine treatment reduced the level of γ-globin gene methylation in a dose dependant manner to a similar extent in each subpopulation except the CD34+CD36− subpopulation that exhibited only minor reduction in the γ-globin gene methylation. These results demonstrate that decitabine treatment demethylates the γ-globin gene primarily in late erythroid progenitors (CD34+CD36+) and erythroid precursors (CD34−CD36+). Methylation of the γ-globin gene is not significantly reduced in the more primitive CD34+CD36- subpopulation after decitabine treatment. The greater sensitivity of the progenitor/precursor subpopulations may be due to increased cell cycle kinetics. The increased levels of DNA methytransferase in CD34+ cells may also contribute to the relative insensitivity of the most primitive subpopulation to decitabine. This analysis identifies the late progenitor/precursor subpopulation as the target subpopulation most sensitive to DNA demethylation by decitabine while the early progenitor/stem cell subpopulation is insensitive to the drug. Table 1: DNA methylation (%) of the γ-globin gene in purified cells of fetal liver and pre- and post-decitabine treated adult bone marrow samples Samples CD34+CD36− CD34+CD36+ CD34−CD36+ Erythroblasts Note: Decitabine doses for PA6973=0.52mg; PA6974=0.26mg; PA7002=0.17mg/kg/day Fetal liver (n=2) 95.4±3.96 66.25±4.17 27.3±1.41 3.7±5.23 ABM-pretreated (n=3) 96.23±0.48 87.21±5.96 79.59±13.42 74.87±8.87 BM-post treated PA6973 85.40 41.30 31.10 37.80 BM-post treated PA6974 94.83 61.90 50.79 52.46 BM-post treated PA7002 92.31 71.93 66.00 58.00

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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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