EKLF Is Recruited to the γ-Globin Gene Promoter as a Co-Activator and Is Required for γ-Globin Gene Induction by Short-Chain Fatty Acids.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1771-1771
Author(s):  
Susan P. Perrine ◽  
Rishikesh Mankidy ◽  
Michael S. Boosalis ◽  
James J. Bieker ◽  
Douglas V. Faller
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Induction ◽  
Therapeutic Interventions ◽  
Short Chain ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.

scholarly journals Short-chain fatty acids induce γ-globin gene expression by displacement of a HDAC3-NCoR repressor complex

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 3179-3186 ◽  
Author(s):  
Rishikesh Mankidy ◽  
Douglas V. Faller ◽  
Rodwell Mabaera ◽  
Christopher H. Lowrey ◽  
Michael S. Boosalis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Globin Gene ◽  
Fatty Acid Derivative ◽  
Gene Promoter ◽  
Adaptor Protein ◽  
Short Chain ◽  
Gene Promoters ◽  
Erythroid Progenitors ◽  
Globin Gene Expression

Abstract High-level induction of fetal (γ) globin gene expression for therapy of β-hemoglobinopathies likely requires local chromatin modification and dissociation of repressor complexes for γ-globin promoter activation. A novel γ-globin–inducing short-chain fatty acid derivative (SCFAD), RB7, which was identified through computational modeling, produced a 6-fold induction in a reporter assay that detects only strong inducers of the γ-globin gene promoter and in cultured human erythroid progenitors. To elucidate the molecular mechanisms used by high-potency SCFADs, chromatin immunoprecipitation (ChIP) assays performed at the human γ- and β-globin gene promoters in GM979 cells and in erythroid progenitors demonstrate that RB7 and butyrate induce dissociation of HDAC3 (but not HDAC1 or HDAC2) and its adaptor protein NCoR, specifically from the γ-globin gene promoter. A coincident and proportional recruitment of RNA polymerase II to the γ-globin gene promoter was observed with exposure to these γ-globin inducers. Knockdown of HDAC3 by siRNA induced transcription of the γ-globin gene promoter, demonstrating that displacement of HDAC3 from the γ-globin gene promoter by the SCFAD is sufficient to induce γ-globin gene expression. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes associated with SCFAD-induced activation of the γ-globin gene and provide a specific molecular target for potential therapeutic intervention.

A Novel Model of Short Chain Fatty Acid (SCFA)- Mediated up-Regulation of Embryonic/Fetal Globin Genes during Definitive Erythropoiesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1878-1878
Author(s):  
Himanshu Bhatia ◽  
Jennifer L. Hallock ◽  
Lauren E. Sterner ◽  
Shay Karkashon ◽  
Amrita Dutta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kinase Activity ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Short Chain ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Basal Media ◽  
Globin Gene Expression

Abstract Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders such as sickle cell disease and β-thalassemia. Short chain fatty acids (SCFAs) up-regulate embryonic/fetal globin gene expression in vitro and in vivo and have great therapeutic potential. We first studied the SCFA-responsiveness of embryonic and fetal globin gene expression during short-term culture in a murine primary cell model. Pooled erythroid fetal liver cells (eFLCs) from wild-type (wt) and human β-globin gene locus-containing mice were examined after treatment with SCFAs. Cells were cultured in basal media alone or in basal media plus propionate (5mm), butyrate (1mM), and/or insulin (2 U/mL) and erythropoietin (10 mg/mL, ‘ins/EPO’). Murine embryonic β-type globin gene expression, ((βH1+εY)/( βH1+εY+βMAJ)x100) was markedly increased at 72 hours in SCFA-treated wt eFLCs, from 1.7±1.2% at baseline to, at 72 hours, 4.88±2.21% in propionate, 5.40±3.39% in butyrate, 19.48±8.30% in butyrate & ins/EPO, compared with ins/EPO-only-treated wt eFLCs, at 0.3±0.3% (n ≥ 3 for each, p<.05). Human fetal γ-globin gene expression was also up-regulated in human transgenic eFLCs, at 29.0±5.0% in butyrate & ins/EPO vs. 3.07±1.08 in ins/EPO only (n=2, p<.05). PCR analysis of FACS-sorted individual eFLCs after culture confirmed that both ins/EPO- and butyrate & ins/EPO-treated cells express detectable α-globin (n=15 for both, p= n.s.). However, only butyrate & ins/EPO-treated cells also express detectable embryonic βH1 globin (11/15, p<.005). Apoptosis was not increased in SCFA-treated eFLCs, with or without ins/EPO. Erythroid differentiation was analyzed by FACS quantitation of CD71 and TER119 co-immunostaining. At 72 hours, 21.5±3.5% of ins/EPO- vs. 65.5±9.9% of butyrate-only- and 77.5±4.0% of propionateonly-treated erythroid cells were highly differentiated (p<.005). Cytospins confirmed a marked increase in erythroid differentiation in SCFA-treated eFLCs. We also investigated SCFA-associated phosphorylation of signaling molecules, notably STAT5 and p38, which had been described in other models of erythropoiesis. Western blotting of protein extracts from cultured eFLCs showed phosphorylation of STAT5 in ins/EPO- and butyrate & ins/EPO-, but not in butyrate-only-, treated cells. However, p38 was constitutively phosphorylated in all of these conditions. Inhibitors of p38 kinase activity, SB 203580 (5–100 μM) or PD 169316 (5–300 μM), added to each condition throughout culture, prevented SCFA-mediated induction of embryonic globin gene expression in a concentration dependent manner. Erythroid differentiation was also blocked by p38 inhibition. Our data suggest that SCFAs augment both pancellular embryonic globin gene expression and erythroid differentiation. However, in our model, p38 phosphorylation and kinase activity are not unique to SCFA treatment in eFLCs; p38-associated erythroid differentiation, common to eFLCs in all culture conditions tested, may be an essential precursor for additional, as yet uncharacterized, molecular triggers for SCFA-mediated reactivation of embryonic/fetal globin gene expression in eFLCs.

scholarly journals Bradykinin stimulation of nitric oxide production is not sufficient for gamma-globin induction

2014 ◽  
Vol 142 (3-4) ◽  
pp. 189-196 ◽  
Author(s):  
Vladan Cokic ◽  
Tijana Suboticki ◽  
Bojana Beleslin-Cokic ◽  
Milos Diklic ◽  
Pavle Milenkovic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Globin Gene ◽  
No Production ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Globin Gene Expression ◽  
Stimulation Of

Introduction. Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases ?-globin and fetal hemoglobin levels in human erythroid progenitors. Objective. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase ?-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 ?M up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/?-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase ?-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce ?/? globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce ?-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of ?-globin gene expression.

Alterations in Protein-DNA Interactions in the γ-Globin Gene Promoter in Response to Butyrate Therapy

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2924-2933 ◽  
Author(s):  
Tohru Ikuta ◽  
Yuet Wai Kan ◽  
Paul S. Swerdlow ◽  
Douglas V. Faller ◽  
Susan P. Perrine
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Dna Interactions ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Protein Dna Interactions ◽  
American Society ◽  
Globin Gene Expression

Abstract The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

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.

LCR 5′HS3 Displays Specificity for ε-Globin Gene Activation during Primitive Erythropoiesis and γ-Globin Gene Activation during Fetal Definitive Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1213-1213
Author(s):  
Kenneth R. Peterson ◽  
Halyna Fedosyuk ◽  
Susanna Harju
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Activation ◽  
Gene Promoter ◽  
Site Specificity ◽  
A Site ◽  
Primitive Erythropoiesis ◽  
Definitive Erythropoiesis ◽  
Globin Gene Expression ◽  
Specific Sequences

Abstract A 2.9 Kb deletion of 5′HS3 (Δ5′HS3) or a 234 bp deletion of the 5′HS3 core (Δ5′HS3c) in a 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) abrogate ε-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that HS sequences of the LCR are involved directly in ε-globin gene activation. The reduction of ε-globin gene transcription in Δ5′HS3 or Δ5′HS3c β-YAC transgenics can be explained by two hypotheses. The first is site-specificity. The interaction between the LCR and the ε-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the ε-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and ε-globin gene transcription is diminished. The second hypothesis is change in conformation of the LCR. Normally, in the embryonic stage, the LCR achieves a three-dimensional conformation that favors interaction with the first gene in the complex, i.e., the ε-globin gene. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, the LCR interacts with the next gene in order, the γ-globin gene. In Δ5′HS3c β-YAC mice, γ-globin gene expression is normal during primitive erythropoiesis, but is extinguished in the fetal stage of definitive erythropoiesis. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not. Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core. To distinguish between these possibilities, β-YAC lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to either an intact LCR, a 2.9 Kb 5′HS3 deletion or a 234 bp 5′HS3 core deletion. Δ5′HS3c Δε::βm β-YAC mice expressed βm-globin throughout development beginning at day 10 in the yolk sac. γ-globin was expressed in the embryonic yolk sac, but not in the fetal liver. Some wild-type β-globin was expressed in addition to βm-globin in adult mice. The γ-globin phenotype is consistent with published data on Δ5′HS3c β-YAC mice. Although ε-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δε::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for ε-globin expression during embryonic erythropoiesis, but not for βm-globin expression. These data support a site specificity model of LCR HS-globin gene interaction. In addition, nuclear ligation experiments provided evidence for a specific physical interaction between 5′HS3 and the γ-globin promoter during fetal definitive erythropoiesis, further supporting a site specificity model.

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 >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.

Gene Expression Profiles Involved in γ to β Globin Gene Switching during Erythroid Maturation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 820-820
Author(s):  
Wei Li ◽  
Betty S. Pace
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Globin Gene ◽  
Gene Expression Profiles ◽  
Phase System ◽  
Mrna Levels ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Gene Switching ◽  
Globin Gene Expression

Abstract The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of hemoglobin accumulation during erythropoiesis and sequential globin gene expression (ε → Gγ → Aγ → δ → β) during development. To gain insights into globin gene switching, we completed time course micorarray analyses of erythroid progenitors to identify trans-factors involved in γ gene activation. Studies were completed to map the pattern of γ and β globin gene expression in progenitors grown from normal peripheral blood mononuclear cells. We compared cells grown in a 2-phase (phase 1, d0-6: SCF, IL-3, IL-6, and GM-CSF and phase 2, d7-25: SCF and EPO) vs. 1-phase (d0-34: SCF, IL-3, and EPO) liquid culture system. From day 0 to 34 in either system cell viability remained >99%. Total RNA was isolated using Trizol and column cleanup (Qiagen). Globin mRNA levels were measured at 2–3 day intervals by quantitative PCR (qPCR). In the 2-phase system γ-globin mRNA>β-globin mRNA up to d14, 4 days of approximately equal expression then β mRNA > γ mRNA by d20. By contrast, in 1-phase studies there was a rapid switch around d20(see graph). We speculate that this difference may be due to the early addition of EPO on d0 therefore we continued our detailed analysis in this system. To confirm that our in vitro system recapitulates in vivo gene expression patterns, we completed studies to ascertain Gγ - vs. Aγ globin mRNA levels. The normalized Gγ:Aγ ratio decreased from ~3:1 on d7 to ~1:1 by d34; These findings were confirmed using two sets of Gγ and Aγ globin primers. We concluded that the 1-phase system recapitulated normal γ/β globin switching and that gene profiling studies to identify the trans-factor involved in switching mechanisms were feasible. We used Discover oligo chips (ArrayIt, Sunnyvale, CA) containing 380 human genes selected from 30 major functional groups including hematopoiesis. To aide interpretation of chip data, cell populations were rated morphologically using Giemsa stained cytospin preps. From d16 on we observed an increase in late erythroid progenitors (normoblasts) from 1% to 71% by d31. After verifying RNA quality by gel inspection of ribosomal molecules, we prepared Cy3 and Cy5 probes for early and late time-point RNA samples respectively. Chip analysis was performed at several time points but d0/21, d7/21, and d21/28 were most informative. Based on Axon GenePixPro 6.0 and Acuity 4.0 software analysis we found the following genes with >1.5-fold change in expression profile (shown as down-regulated/up-regulated genes): d0/21: 33/73, d7/21: 13/25, and d21/28:35/26. Principal component analysis (PCA), hierarchical clusters and self organizing maps were constructed. Gene profiles were correlated with the γ/β switching curve using d7 (γ >β), d21 (γ ~ β), and d28 (γ <β) data. Hematopoietic dataset analysis at d21 revealed 4 candidate γ-globin gene activators including v-myb, upsteam binding transfactor -RNApol1 and 2 zinc finger proteins. Analysis of a d28 dataset revealed 12 proteins involved in γ-globin gene silencing including IL-3, SCF, MAPKKK3, v-raf-1, ATF-2, and glucocorticoid receptor DNA binding factor 1 among others. Gene expression profiles will be validated using qPCR and promising candidates will be tested by forced expression in transient and stable reporter systems. Figure Figure

Cucurbitacin D Upregulates Fetal Hemoglobin Expression in K562 Cells and Human Erythroid Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3833-3833
Author(s):  
Hongtao Xing ◽  
Siwei Zhang ◽  
H. Phillip Koeffler ◽  
Ming Chiu Fung
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Control ◽  
Optimal Dosage ◽  
Erythroid Progenitors ◽  
Cucurbitacin D ◽  
Globin Gene Expression ◽  
Sodium Phenylbutyrate

Abstract The search for novel therapeutic candidates causing reactivation of fetal hemoglobin (a2g2; HbF) to reduce the imbalance of globin gene expression is important in order to develop effective approach for the clinical management of sickle cell anemia and b-thalassemia. For the first time, we have identified cucurbitacin D (CuD), a naturally occurring oxygenated tetracyclic triterpenoid, as a molecular entity inducing g-globin gene expression and HbF synthesis in K562 cells and human erythroid progenitors from either peripheral blood or bone marrow. The upregulation of HbF induced by CuD was dose- and time- dependent. CuD was compared to hydroxyurea (HU), 5-azacytidine, amifostine, recombinant human erythropoietin (rhEPO), and sodium phenylbutyrate. At their optimal dosage, CuD (12.5 ng/mL) and HU (25.0 μg/mL) induced nearly 70% K562 cells to express total hemoglobin after 6 days culture, which was higher than the induction by Amifostine (30%), 5-azacytidine (36%), rhEPO (16%), sodium phenylbutyrate (23%) at their optimal concentrations and negative control (11%). Fetal hemoglobin ELISA showed that CuD (12.5 ng/mL) and 5-azacytidine (400 ng/mL) induced higher levels of fetal hemoglobin in K562 cells (15.4 ng/μL and 29.3 ng/μL, respectively), compared to HU (10.3 ng/μL), amifostine (7.8 ng/μL), rhEPO (10.9 ng/μL), sodium phenylbutyrate (9.9 ng/μL) at their optimal concentrations and negative control (5.3 ng/μL). CuD induced a significantly higher fetal cell percentage than HU in K562 cells (65% vs 37% maximum) and primary erythroid progenitors (36% vs 21% maximum) based on the immunofluorescence imaging and flow cytometry analysis. Real-time PCR results showed that the amount of γ-globin mRNA increased from 2.5-fold in CuD-optimal-treated cells (12.5 ng/mL, 48 hours) compared with 1.5-fold in HU-optimal-treated cells (25.0 μg/mL, 48 hours). Growth inhibition assay (MTT) demonstrated that CuD at its optimal γ-globin inducing dosage (12.5 ng/mL) inhibited proliferation of K562 by less than 10% of untreated control cells; while hydroxyurea at its optimal dosage (25.0 μg/mL) inhibited 80% of cell division. The in vitro therapeutic index (calculated by dividing the dose inhibiting 50% cell growth (IC50) by dose inducing 50% maximal HbF production (ED50)) of CuD was 40-fold greater than HU. Taken together, the results suggest that CuD has the potential to be a therapeutic agent for treatment of sickle cell anemia and b-thalassemia.

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