scholarly journals Restoration of the CCAAT Box or Insertion of the CACCC Motif Activate δ-Globin Gene Expression

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 421-427 ◽  
Author(s):  
Delia C. Tang ◽  
David Ebb ◽  
Ross C. Hardison ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Globin Gene ◽  
Treatment Strategies ◽  
K562 Cells ◽  
Promoter Sequence ◽  
Flanking Sequence ◽  
Globin Promoter ◽  
Ccaat Box ◽  
Globin Gene Expression

Abstract Hemoglobin A2 (HbA2 ), which contains δ-globin as its non–α-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the δ-globin gene in adult erythroid cells, we first compared promoter sequences and found that the δ-globin gene differs from the β-globin gene in the absence of an erythroid Krüppel-like factor (EKLF ) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human δ-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5′ flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the δ promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type δ promoter. A set of substitutions that create an EKLF binding site centered at −85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase δ-globin gene expression, with potential future clinical benefit.

scholarly journals Restoration of the CCAAT Box or Insertion of the CACCC Motif Activate δ-Globin Gene Expression

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 421-427 ◽  
Author(s):  
Delia C. Tang ◽  
David Ebb ◽  
Ross C. Hardison ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Globin Gene ◽  
Treatment Strategies ◽  
K562 Cells ◽  
Promoter Sequence ◽  
Flanking Sequence ◽  
Globin Promoter ◽  
Ccaat Box ◽  
Globin Gene Expression

Hemoglobin A2 (HbA2 ), which contains δ-globin as its non–α-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the δ-globin gene in adult erythroid cells, we first compared promoter sequences and found that the δ-globin gene differs from the β-globin gene in the absence of an erythroid Krüppel-like factor (EKLF ) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human δ-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5′ flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the δ promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type δ promoter. A set of substitutions that create an EKLF binding site centered at −85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase δ-globin gene expression, with potential future clinical benefit.

scholarly journals Activation of β-Globin Promoter by Erythroid Krüppel-Like Factor

1998 ◽  
Vol 18 (1) ◽  
pp. 102-109 ◽  
Author(s):  
Haruhiko Asano ◽  
George Stamatoyannopoulos
Keyword(s):  
Binding Site ◽  
Zinc Finger Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Sequence Difference ◽  
Optimal Position ◽  
Finger Protein ◽  
Primary Determinant ◽  
Globin Promoter ◽  
Globin Gene Expression

ABSTRACT Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the β-globin gene CACCC box and is essential for β-globin gene expression. EKLF does not activate the γ gene, the CACCC sequence of which differs from that of the β gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the β gene by EKLF, the CACCC boxes of β and γ genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the β promoter carrying a γ CACCC box at a level comparable to that at which it activated the wild-type β promoter, whereas EKLF failed to activate a γ promoter carrying the β CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the β promoter carrying the γ CACCC box, or that EKLF activated the mutated β promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the β or γ promoter determined EKLF specificity, the proximal β CACCC box sequence was created at the position of the β promoter (−140) which corresponds to the position of the CACCC box on the γ promoter. Similarly, the β CACCC box was created in the position of the γ promoter (−90) corresponding to the position of the CACCC box in the β promoter. EKLF retained weak activation potential on the β−140CAC promoter, whereas EKLF failed to activate the γ−90βCAC promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the β promoter by EKLF is determined by the overall structure of the β promoter rather than solely by the sequence of the β gene CACCC box.

Characterization of Histone Deacetyalses Involved in γ-Globin Gene Regulation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1869-1869
Author(s):  
Shalini A Muralidhar ◽  
Sadeieh Nimer ◽  
Betty Pace
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Promoter Activity ◽  
Globin Gene ◽  
Indirect Evidence ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Globin Promoter ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

Abstract Fetal hemoglobin (HbF; α2γ2) ameliorates vaso-occlusive symptoms in individuals with sickle cell disease (SCD) because of its ability to inhibit hemoglobin S polymerization. One mechanism for γ-globin reactivation likely involves chromatin modification and the release of repressor complexes in which histone deacetylases (HDACs) may be present. The objective of this study was to identify HDACs involved in γ-globin gene regulation. Experiments were performed in K562 cells to determine the ability of NaB (2mM), TSA (0.5μm) and the non-HDAC-inhibitor γ-globin activator hemin (50μM), to alter transcription levels of the HDAC genes during concomitant HbF induction. Gene expression was measured by reverse transcription (RT) of mRNA followed by quantitative PCR (qPCR) analysis using gene-specific primers. Treatment of K562 cells with TSA and NaB reduced transcription levels of both HDAC9 and HDRP (histone deacetylase related protein, a splice variant of HDAC9) from 20–80% as did the control agent hemin (p&lt;0.05). By contrast, expression of HDAC7 and HDAC10 was enhanced in the presence of both HDAC inhibitors. The altered HDAC gene expression levels provided indirect evidence for a possible role in mechanisms of γ-globin response to drug inducers. Subsequent experiments were performed to delineate whether HDAC9 and HDRP are directly involved in γ-globin regulation. We performed siRNA knockdown of HDAC9 and HDRP in K562 cells to determine the effect on expression of endogenous γ-globin. siRNA oligonucleotides were transfected using Oligofectamine (Invitrogen) for 48 hrs and expression of targeted genes were quantified by RT-qPCR. siHDAC9 and siHDRP (Dharmacon) treatment resulted in dose-dependent γ-globin silencing and transactivation respectively at 80–320nM. Experiments were then performed with 160nM of siHDAC9 or siHDRP in the K562 cell lines which were stably transfected with a luciferase reporter (pGL4.17-Luc2-neo, Promega) under the control of Gγ-globin promoter (−1500 to +36) and the pGL4.17-Luc2-neo empty vector. We likewise observed a 30% decrease in luciferase activity with siHDAC9 and a 40% increase with siHDRP suggesting that HDAC9 and its variant are directly involved in regulation of γ-promoter activity. In summary, the effects on endogenous γ globin levels and Gγ globin promoter activity through HDAC9/HDRPspecific knockdown by siRNA experiments suggest that HDAC9 molecules play a role in regulating γ-globin gene expression. We conclude that HDAC9 and HDRP have opposite regulatory effect on γ-globin gene expression and may act by a feedback mechanism.

Induction of Fetal Hemoglobin by Transcriptional Co-Activators MTF-1 and TSPYL1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 353-353 ◽  
Author(s):  
Kenneth R Peterson ◽  
Flavia C Costa ◽  
Halyna Fedosyuk ◽  
Renee Neades ◽  
Johana Bravo de los Rios ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Complex ◽  
Globin Gene ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Zinc Metabolism ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract Abstract 353 Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the γ-globin gene-promoters via binding of the GATA-2 protein. In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures: No relevant conflicts of interest to declare.

SATB1 family protein expressed during early erythroid differentiation modifies globin gene expression

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3330-3339 ◽  
Author(s):  
Jie Wen ◽  
Suming Huang ◽  
Heather Rogers ◽  
Liliane A. Dickinson ◽  
Terumi Kohwi-Shigematsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Family Protein ◽  
Globin Promoter ◽  
Globin Gene Expression

AbstractSpecial AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with ϵ-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases γ-globin and down-regulates SATB1 family protein and ϵ-globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the β-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the ϵ-globin promoter. SATB1 overexpression increases ϵ-globin and decreases γ-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the ϵ-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the γ-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in ϵ-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the ϵ-globin gene by interaction with specific sites in the β-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation. (Blood. 2005;105:3330-3339)

Induction of γ-Globin Gene Expression Via the Nrf2/Antioxidant Response Element Signaling Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 975-975
Author(s):  
Elizabeth Macari ◽  
Rachel West ◽  
William J. Lowrey ◽  
Rodwell Mabaera ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Globin Gene ◽  
K562 Cells ◽  
Antioxidant Response ◽  
Antioxidant Response Element ◽  
Globin Mrna ◽  
Nrf2 Pathway ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract Abstract 975 Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including HU, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoeisis and the possibility of long-term side effects including those related to DNA mutation and epigenetic changes. Recently, two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemoprevention agents. These and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor NF-E2 related factor 2 (Nrf2) which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between two CAAT boxes, suggesting that it too may be activated by Nrf2. This led us to hypothesize that drugs which activate the ARE/Nrf2 pathway may provide a less toxic approach to HbF induction. Several compounds that activate this pathway are already approved for human use or are under investigation in human trials. To test this hypothesis, we treated K562 cells with various Nrf2 pathway activators. First, we determined the maximum concentration of each compound that did not inhibit proliferation of K562 cells. Using these doses, we performed time course experiments by treating K562 cells and then measuring steady-state mRNA levels of Nrf2 target genes and γ-globin using quantitative real-time PCR. We initially tested six compounds that are known to induce antioxidant response genes. We found the most pronounced γ-globin induction followed treatment with tert-butylhydroquinone (tBHQ) (2.8 fold), 3H-1,2 dithiole-3-thione (D3T) (2.4 fold) and curcumin (2.1 fold). We next tested tBHQ in erythroid precursors isolated from normal human bone marrow. In these primary cells it also significantly increased expression of γ-globin mRNA and of Nrf2 target genes NQO1, GCLM and GSTP1. To determine if Nrf2 was necessary for tBHQ induction of γ-globin mRNA we used siRNA to knockdown Nrf2 expression in K562 cells. Our results show that transiently silencing Nrf2 transcription prevented γ-globin and NQO1 gene induction by tBHQ compared to samples transfected with scrambled siRNA (p < 0.01). To determine if this requirement for Nrf2 is due to Nrf2 binding at the ARE consensus sequence in the γ-globin promoter, ChIP analysis was performed. This demonstrated that Nrf2 binding at both the γ-globin and NQO1 promoters was progressively increased at 4 and 6 hours after tBHQ treatment compared to the negative control necdin promoter where there was no change. Taken together, these results suggest that the antioxidant tBHQ induces γ-globin mRNA expression through the Nrf2/ARE pathway and that this may be a less toxic strategy for γ-globin gene induction in people with hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

Regulation of the Human α-Globin Genes by GKLF.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1867-1867
Author(s):  
Paolo Moi ◽  
Giuseppina Maria Marini ◽  
Loredana Porcu ◽  
Isadora Asunis ◽  
Maria Giuseppina Loi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Point Mutations ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Globin Genes ◽  
Small Interfering Rnas ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β- and β-like globin genes. Prompted by the observation that four KLF sites are distributed in the human α-globin promoter, we investigated if any of the β-globin cluster regulating KLFs could also act to modulate the expression of the α-globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a α-globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous α-globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the α-globin genes. All experiments combined support a main regulatory role of GKLF in the control of α-globin gene expression.

scholarly journals Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 738-746 ◽  
Author(s):  
BL Tonkonow ◽  
R Hoffman ◽  
D Burger ◽  
JT Elder ◽  
EM Mazur ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Globin Gene ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Globin Mrna ◽  
Human Leukemia Cell Line ◽  
Globin Gene Expression

Abstract The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3–4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3–5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Subdomains of the Baboon (P. anubis) β-Globin Gene Cluster Are Differentially Sensitive to Dacogen Treatment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 525-525
Author(s):  
Janet Chin ◽  
Donald Lavelle ◽  
Bryan Roxas ◽  
Kestis Vaitkus ◽  
Maria Hankewych ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Intergenic Region ◽  
Globin Gene ◽  
Globin Genes ◽  
Gene Complex ◽  
Alu Sequence ◽  
Low Levels ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract Understanding the mechanism responsible for the developmental regulation of the β-like globin genes would be important in the design of future pharmacologic therapies to increase fetal hemoglobin (HbF) in patients with sickle cell disease and β-thalassemia. The baboon is a valuable and relevant experimental animal model to study the regulation of globin gene expression because the structure of the β-globin gene complex and developmental pattern of globin gene expression are similar to human, and HbF levels are greatly increased following treatment of baboons with the DNA methyltransferase inhibitor Dacogen (5-aza-2′-deoxycytidine; DAC). To investigate the relationship between DNA methylation, chromatin structure and globin gene expression, the pattern of acetylated histone H3 (ac-H3) and H4 (ac-H4) within the β-globin gene complex was compared in purified erythroblasts from baboon fetal liver (FL; n=2) and bone marrow (ABM; n=2) of adult baboons pre and post DAC treatment. HbF increased to high levels (67.8%, 61.9%) in respective animals and methylation of 18 CpG sites within the ε- and γ globin genes was reduced &gt;50% following DAC treatment. Enrichment of ac-H3 and ac-H4 throughout the β-globin gene complex was measured by chromatin immunoprecipitation (ChIP) followed by real time PCR. In FL, equivalent levels of ac-H3 and ac-H4 were observed near the ε-globin and γ-globin promoters that were 3 fold higher than near the Aγ-enhancer and pseudo-β gene and 5–14 fold higher than near the β-globin promoter. In pretreatment ABM, levels of ac-H3 and ac-H4 near the β-globin promoter were 4–6 fold greater than near the γ-globin promoter, Aγ-enhancer, and pseudo-β gene and 10-15 fold higher than near the ε-globin promoter. The lowest levels of histone acetylation were observed in a 6kb subdomain within the γ-β intergenic region extending from the duplicated Alu sequence to 3′ of the δ-globin gene. Following DAC treatment, histone acetylation of the ε-, γ-, and pseudo-β genes and Aγ-enhancer increased 4-10 fold, while histone acetylation of the β-globin gene remained unchanged. This resulted in equivalent levels of histone acetylation associated with the γ-globin gene, Aγ-enhancer, pseudo-β-, and β-globin genes that were 3 fold greater than with the ε-globin gene. The levels of histone acetylation within the 6 kb subdomain of the γ-β intergenic region remained low. Our results suggest that three subdomains of chromatin are present within the baboon β-globin gene complex. One subdomain that encompasses the ε-, γ-, and pseudo-β genes is characterized by high levels of histone acetylation in FL and low levels in ABM. DAC treatment increases histone acetylation within this region to levels observed near the β-globin gene. A second subdomain near the β-globin gene is characterized by high levels of histone acetylation in ABM and low levels in FL. Histone acetylation of the β-globin gene within this subdomain remains high following DAC. A third subdomain found within the γ-β intergenic region surrounding the duplicated Alu sequences is characterized by a low level of histone acetylation in both FL and ABM. The level of histone acetylation of this region remains low following DAC. We conclude that three chromatin subdomains within the β-globin gene complex are differentially sensitive to DAC-induced changes in histone acetylation.

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