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

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

Blood ◽

10.1182/blood.v122.21.438.438 ◽

2013 ◽

Vol 122 (21) ◽

pp. 438-438

Author(s):

Maria Armila Ruiz ◽

Angela Rivers ◽

Kestis Vaitkus ◽

Tatiana Kouznetsova ◽

Nadim Mahmud ◽

...

Keyword(s):

Dna Methylation ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Erythroid Differentiation ◽

Dna Demethylation ◽

Dna Hypomethylation ◽

Liquid Cultures ◽

Erythroid Precursors ◽

Globin Promoter

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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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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Lenalidomide and Novel Immunomodulatory Drugs (IMiDs®): A New Approach to the Regulation of Erythropoiesis and Hemoglobin Synthesis in Anemia.

Blood ◽

10.1182/blood.v106.11.2702.2702 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2702-2702 ◽

Cited By ~ 2

Author(s):

Laure Moutouh de Parseval ◽

Helen Brady ◽

Dominique Verhelle ◽

Laura G. Corral ◽

Emilia Glezer ◽

...

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Chemotherapeutic Agents ◽

Immunomodulatory Drugs ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Hemoglobin Synthesis ◽

Hematological Cancers

Abstract Clinical trial results have demonstrated that lenalidomide (Revlimid®) reduces or even eliminates the need for red blood cell transfusions in some anemic myelodysplastic patients. We have examined whether lenalidomide and Actimid™, members of a new class of immunomodulatory drugs (IMiDs®), which are currently under evaluation for the treatment of hematological cancers could regulate erythropoiesis and hemoglobin synthesis. For this purpose, we used an in vitro culture model to differentiate human erythroid progenitors from bone marrow or peripheral blood CD34+ cells. We demonstrate that lenalidomide and AztimidTM modulate erythropoiesis and increase proliferation of immature erythroid cells. In addition to the regulation of erythroid differentiation, lenalidomide and ActimidTM are potent inducers of fetal hemoglobin. Unlike other inducers of fetal hemoglobin such as 5-aza-cytidine that are cytotoxic, IMiDs® promoted survival of erythroblast cultured with known cytotoxic drug. Gene expression profiling of erythroid differentiated cells showed that IMiDs® regulate specific erythroid transcription factors and genes that participate in hemoglobin synthesis, and genes invoved in cell cycle and cellular differentiation. Globin gene expression is controlled by IMiDs® during erythroid differentiation by inducing fetal hemoglobin synthesis. Our results support the hypothesis that IMiDs® restore effective erythropoiesis in myelodysplastic patients and protect erythroid cells from the cytotoxic effect of chemotherapeutic agents. In conclusion, IMiDs® may represent an interesting new therapy for cancer-related anemia and β-hemoglobinopathies.

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TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽

10.1182/blood.v124.21.2912.2912 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2912-2912

Author(s):

Petros Papadopoulos ◽

Laura Gutierrez ◽

Jeroen Demmers ◽

Dimitris Papageorgiou ◽

Elena Karkoulia ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fetal Liver Cells ◽

Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

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siRNA Targeting DNA Methyltransferase I Increases ε- and γ-Globin Expression.

Blood ◽

10.1182/blood.v114.22.973.973 ◽

2009 ◽

Vol 114 (22) ◽

pp. 973-973

Author(s):

Virryan Banzon ◽

Vinzon Ibanez ◽

Kestis Vaitkus ◽

Kenneth Peterson ◽

Joseph DeSimone ◽

...

Keyword(s):

Dna Methylation ◽

Real Time ◽

Real Time Pcr ◽

Dna Methyltransferase ◽

Globin Gene ◽

Pcr Analysis ◽

Globin Promoter ◽

Chain Synthesis ◽

Globin Chain Synthesis ◽

In Cells

Abstract Abstract 973 Pharmacological inhibitors of DNA methyltransferase (DNMT) increase fetal hemoglobin (HbF) levels in experimental primates and patients with sickle cell disease. Therefore we hypothesize that DNMT is directly involved in maintaining repression of the γ-globin gene in adult stage erythroid cells. To test this hypothesis, levels of DNMT1 in mouse chemical-of-dimerization (CID) bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and primary cultured erythroid progenitor cells (EPC) derived from baboon CD34+ BM cells were reduced by treatment with siRNA targeting DNMT1 (siDNMT1) and the effect on globin gene expression determined. Nucleofection conditions that achieved 80-90% transfection efficiency were used to introduce siRNA into CID-dependent mouse βYAC BM cells. Real time PCR analysis showed that expression of DNMT1 was decreased 70-80% in cells treated with siDNMT1 compared to cells transfected with nonsilencing control siRNA while DNMT3A and 3B were not decreased. Results of real time PCR analysis of six independent experiments showed that ε-globin expression was increased 65.3+/−37.8 fold, γ-globin 230.3+/−147.8 fold, and β-globin 6.0+/−3.3 fold in cells treated with siDNMT1 compared to untreated controls, while cells treated with nonsilencing siRNA showed minimal (<2 fold) changes. The difference in ε- and γ-globin expression between cells treated with siDNMT1 and nonsilencing RNA was significant (p<.01). Bisulfite sequence analysis showed that DNA methylation of the ε-globin promoter and γ-globin promoters were reduced to 25.7 and 53.3% dmC, respectively, in cells treated with siDNMT1 compared to 68.8 and 98.8% dmC, respectively, in cells treated with nonsilencing siRNA. Nucleofection of cultured baboon EPC with siDNMT1 or nonsilencing siRNA was performed on d7 and d8 of culture. Transfection efficiencies of 45-50 % were achieved. Expression of DNMT1 was decreased >80% in cells treated with siDNMT1 compared to those treated with nonsilencing siRNA. Real time PCR analysis of duplicate samples showed that γ-globin expression was increased 2.06 and 2.25 fold relative to untreated controls following treatment of cells with siDNMT1 while nonsilencing siRNA had no effect. Expression of ε-globin was increased 35.26 and 25.4 fold relative to untreated controls in cells treated with siDNMT1 while a lesser effect was observed in cells treated with nonsilencing siRNA (7.41 and 8.16 fold). HPLC analysis of biosynthetically radiolabelled globin chains showed that γ-globin chain synthesis (γ/γ+β ratio) was increased in cells treated with siDNMT1 (0.59 and 0.63) compared to cells treated with nonsilencing siRNA (0.41 and 0.37), untreated controls (0.39) and mock-transfected controls (0.43). DNA methylation of 3 CpG residues within the 5' ε-globin promoter region and 5 CpG residues within the 5' γ-globin promoter region was reduced to 52.8 and 45.0% dmC, respectively, in cells treated with siDNMT1, compared to 100 and 85.4% dmC, respectively, in cells treated with nonsilencing siRNA. Our results demonstrate that siDNMT1 reduces DNMT1, reduces levels of DNA methylation of the ε- and γ-globin gene promoters, and increases ε- and γ-globin gene expression and γ-globin chain synthesis in CID-dependent mouse BM cells and in primary baboon EPC cultures derived from CD34+ BM cells. We conclude that DNMT1 is critically involved in the mechanism responsible for repression of γ-globin expression in adult-stage erythroid cells and therefore inhibition of DNMT1 activity by pharmacological inhibitors of DNA methyltransferase likely plays a fundamental role in the ability of these drugs to increase HbF in vivo. 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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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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