scholarly journals Induction of Fetal HemoglobinIn VivoMediated by a Syntheticγ-Globin Zinc Finger Activator

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Flávia C. Costa ◽  
Halyna Fedosyuk ◽  
Renee Neades ◽  
Johana Bravo de Los Rios ◽  
Carlos F. Barbas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zinc Finger ◽  
Yeast Artificial Chromosome ◽  
Bone Marrow Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Proximal Promoter ◽  
Erythroid Progenitor ◽  
Globin Gene Expression

Sickle cell disease (SCD) andβ-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF,γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation ofγ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the −117 region of theAγ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a humanβ-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34+erythroid progenitor cells from normal donors andβ-thalassemia patients. Herein, we report that gg1-VP64 increasedγ-globin gene expressionin vivo, in peripheral blood samples from gg1-VP64β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.

Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 962-962
Author(s):  
Kenneth R Peterson ◽  
Levi C Makala ◽  
Mayuko Takezaki ◽  
Carlos F Barbas ◽  
Betty Pace
Keyword(s):  
Gene Expression ◽  
Zinc Finger ◽  
Sickle Cell ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Proximal Promoter ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Globin Gene Expression

Abstract A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. Disclosures Makala: Georgia Regents University: Employment.

Induction of Human γ Globin Gene Expression by Histone Deacetylase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1583-1583
Author(s):  
Hua Cao
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Hydroxamic Acid ◽  
Globin Gene ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Erythroid Progenitor ◽  
Globin Gene Expression ◽  
Hydroxamic Acid Derivatives

In previous studies we have showed that HDAC inhibitors including hydroxamic acid derivatives of short chain fatty acids butyryl hydroxamate, propionyl hydroxamate, subericbis hydroxamic acid (SBHA), and suberoylanilide hydroxamic acid (SAHA), are potent inducers of γ globin gene expression in in vitro luciferase assays and in cultures of human adult erythroid progenitor cells. In this present study, we used μLCR Aγ transgenic mice to test whether these compounds can also induce γ gene expression in vivo. We found that in addition to γ gene induction these compounds have considerable erythropoiesis activity. Thus, Propionyl and butyryl hydroxamate increased reticulocytes of mice by 71% and 139%, the in vivo BFUe counts by 75% and 51% and the in vivo γ gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. We conclude that Hydroxamic acid derivatives can stimulate both the in vivo erythropoiesis and fetal globin production in a thalassemic murine model. Cyclic depsipeptide FK228 is a highly potent histone deacetylase inhibitor, currently in clinical trials in cancer patients. We investigated whether FK228 also functions as inducer of human γ globin gene expression and compared Hb F induction by FK228 to that of four other HDAC inhibitors, including hydroxamic acids (TSA), synthetic benzamides (MS-275), and two cyclic tetrapeptides, Apicidin and HC-Toxin. Our results showed that FK228 is the most potent fetal hemoglobin inducer among all the HDAC inhibitors tested in our laboratory. In a concentration of 0.84 nanomolar, FK228 induces γ gene promoter activity in the dual luciferase assay by 7.81 fold. In the human erythroid progenitor cell cultures it increases the levels of γ mRNA by 8.48 fold in a concentration of 0.143 nM. In contrast, fetal hemoglobin induction by other HDAC inhibitors is achieved in concentrations that are 100 to 1000 fold higher. We conclude that FK228 is a promising compound for induction of Hb F in patients with sickle cell disease and thalassemia.

O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1020-1020
Author(s):  
Kenneth R Peterson ◽  
Zhen Zhang ◽  
Ee Phie Tan ◽  
Anish Potnis ◽  
Nathan Bushue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sodium Butyrate ◽  
Yeast Artificial Chromosome ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Globin Gene Expression ◽  
Dynamic Cycling

Abstract Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. Disclosures: No relevant conflicts of interest to declare.

Hydroxamic Acids Derivatives Induce γ Globin Gene Expression in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1224-1224
Author(s):  
Hua Cao ◽  
Manfred Jung ◽  
George Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Animal Model ◽  
Hydroxamic Acid ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Hydroxamic Acids ◽  
Rnase Protection ◽  
Acid 500Mg ◽  
Globin Gene Expression

Abstract We have previously shown that four hydroxamic acids: butyric and propionic hydroxamic acids, subericbishydroxamic acid (SBHA) and suberoylanilide hydroxamic acid (SAHA) are potent inhibitors of histone deacetylase and strong inducers of fetal hemoglobin expression in vitro (Exp Hematol.31:197, 2003). In the present study we tested their effect on fetal hemoglobin synthesis in vivo. Transgenic mice carrying the human μLCR Aγ construct continue to express the human γ gene in the adult stage of development ( γ/α mRNA ratio ~ 5%, Blood.77:1326, 1991). These mice were crossed to mice heterozygous for a thalassemia gene due to β globin gene deletion (PNAS.92:11608, 1995). The β thalassemia/μLCR Aγ mice represent an appropriate moderately anemic animal model for testing the effects of Hb F inducers. Compounds were administered subcutaneously with a mini-osmotic pump continuously for 7days in a high and a low concentration. Concentrations were: for butyric hydroxamic acid: 500mg/kg/day/100mg/kg/day; for propionic hydroxamic acid: 500mg/kg/day/100mg/kg/day; for SAHA: 100mg/kg/day/20mg/kg/day; and for SBHA: 200mg/kg/day/40mg/kg/day. Two test groups were studied. In group 1, 70μL mice blood was drawn every other day up to 20 days; in group 2, 70μL mice blood was drawn only on days 0 and 21. Reticulocytes and F reticulocytes were measured using flow cytometry, while γ globin gene expression was quantitated by RNase protection assay. Butyric and propionate hydroxamic acids increased reticulocytes by 70.52% (from 13.96% to 23.81%) and 172.52% (from 10.34% to 28.20%) respectively. There was only small increase in reticulocytes in the mice treated with SAHA (from 13.33% to 15.36%), SBHA (from 14.24% to 16.27%) and the PBS control (11.06% to 14.11%). All the compounds increased the level of γ mRNA: butyric hydroxamate by 53.07%; propionic hydroxamate by 40.05%; SAHA by 49.87%, and SBHA by 34.05%. These results suggest first that all the hydroxamic acid derivatives we used increase fetal hemoglobin in vivo in the thalassemia animal model; second butyric and propionic hydroxanic acids are in addition inducers of in vivo erythropoiesis.

Endogenous Elevations of Short Chain Fatty Acids (SCFAs) Can Up-Regulate Embryonic Globin Gene Expression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1770-1770
Author(s):  
Himanshu Bhatia ◽  
Jennifer Hallock ◽  
Lauren Sterner ◽  
Toru Miyazaki ◽  
Ann Dean ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Short Chain Fatty Acids ◽  
Yolk Sac ◽  
Globin Genes ◽  
Key Enzyme ◽  
Globin Gene Expression

Abstract Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders. Since SCFAs can affect embryonic and fetal globin gene expression, we examined their role during development. Murine globin gene expression, β-type (embryonic βH1, and epsilon-y, εY, and adult βmajor), and alpha (α)-type (embryonic zeta, ζ, >α, adult α), were compared between wildtype (wt) and transgenic mice, in which a key enzyme for SCFA metabolism, PCCA, had been knocked out (PCCA−/−, (Miyazaki et al, 2001). E10.5 PCCA−/− yolk sac (n= 9), showed increased α, βH1 and ζ gene expression, at respectively 2-, 2.6- and 1.6-fold relative to wt (n=13, p<.05), and εY gene expression, at 1.7-fold (p=0.07). The embryonic-to-adult globin gene switch was modestly delayed in yolk sacs from E12.5 PCCA−/− (n=9) vs. wt (n=4) and E 14.5 PCCA−/− (n=6) vs. wt (n=6). % embryonic β-type globin gene expression (% βH1 and εY of total β globin) was 77±6 PCCA−/− and 74±3 wt at E12.5, p=n.s., and 42±13 PCCA−/− and 21±3 wt at E14.5, p<.05; % emvbryonic α-type expression (% ζ of total α) was 32±3 PCCA−/−, 25±1wt at E12.5, p<.05 and 7±2 PCCA−/− and 4±1 wt at E14.5, p<.05). Embryonic globin gene expression in E 12.5 and 14.5 fetal livers was not different between PCCA−/− and wt embryos. Cultures of pooled E14.5 wt fetal liver cells (FLCs, n=4 separate experiments), however, suggested that embryonic globin genes can be activated in FLCs. The percent of total β-type globin gene expression that was embryonic after culture with butyrate (1mM) was 11.6±2.6%, with propionate (2.5 mM) was 3.6±0.2%, and insulin/erythropoietin or basal media was 0.03±0.03% and 0.42±0.26% respectively (p<.05 relative to SCFAs). Dose-response with propionate (n=2 seaparate experiments) suggest inadequate endogenous propionate levels for activation in PCCA −/− fetal liver, as % embryonic β-type globin gene expression rose above basal levels only at concentrations of 1 to 5 mM (2.5 mM maximal) but not at <0.6 mM. We conclude that endogenous SCFAs, at levels achievable in vivo can activate embryonic globin gene expression during development in the murine yolk-sac. However, higher levels than achievable endogenously currently are necessary to produce this effect in murine fetal livers.

A Mutation in a GATA-1 Binding Site 5′ to the Gγ-Globin Gene (nt -567, T>G) May Be Associated with Increased Levels of Fetal Hemoglobin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 500-500 ◽  
Author(s):  
H.Y. Luo ◽  
D. Mang ◽  
G.P. Patrinos ◽  
C.J.Y. Wu ◽  
S.H. Eung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Family Members ◽  
Point Mutations ◽  
Fetal Hemoglobin ◽  
Proximal Promoter ◽  
Globin Genes ◽  
Nucleotide Polymorphisms ◽  
Mobility Shift ◽  
Globin Gene Expression

Abstract The identification of single nucleotide polymorphisms (SNPs) associated with increased fetal hemoglobin (Hb F) levels in adults provide important insights to the regulation of γ-globin gene expression, and the modulation of Hb F production in severe β hemoglobinopathies. Fourteen known point mutations located between nucleotide (nt) −110 to −205 5′ to the Gγ- and Aγ-globin genes are associated with hereditary persistence of fetal hemoglobin (HPFH). These likely affect the interactions between transcription factors and proximal promoter elements. We investigated both parents and 2 sons of an Iranian-American family, in whom the father and his younger son had elevated Hb F levels (See table). The mother and her older son were heterozygous for the (−α3.7) single α-globin gene deletion, likely accounting for their borderline microcytosis. Extensive nt sequencing of the β-globin gene and promoters of Gγ- and Aγ-globin genes was carried out. No known β-thalassemia mutation was detected in any of the 4 family members. None of the known HPFH point mutations was present. The C>T SNP (Xmn I) at nt −158 5′ to the Gγ-globin gene that has been associated with increased Gγ-globin gene expression was also not found. However, a novel T>G substitution was detected at nt −567 5′ to the Gγ-globin gene in the father and his younger son, but not in the mother and her older son. This SNP alters a putative GATA-1 binding sequence, AGATAA to AGAGAA. Haplotyping of the Gγ Aγβ region in the family showed that the T>G SNP in the father and his younger son resides on the same GγAγβ haplotype. This SNP was not present in 15 individuals of diverse racial and ethnic origins, in 186 Thai individuals, and in 133 of 134 Iranians living in Tehran. To our knowledge, this SNP has not been previously reported in the literature. To begin to study the functional significance of this SNP, gel mobility shift analysis was done with two 40 nt long oligomers, one with the wild type GATA sequence and the other with the mutated GAGA sequence, using uninduced mouse erythroleukemia (MEL) cell nuclear extracts. The mutant GAGA sequence results in a complete loss of GATA-1 binding. The region from nt −382 to −730 5′ to the Aγ-globin gene was reported to be related to Aγ-globin gene silencing (Stamatoyannopoulos et al, Mol Cell Biol 13:7636, 1993). The nt −567 T>G SNP is located within the comparable region of the Gγ-globin gene, which is highly homologous to the Aγ-globin gene. Among the 4 family members under study, no other SNPs are found in the same region. Taken together, these observations raise the possibility that the T>G SNP at nt −567 5′ to the Gγ-globin gene is associated with elevated Hb F, that might be caused by a novel mechanism, i.e., incomplete silencing of the Gγ-globin gene, resulting from the abolished GATA-1 binding. Additional clinical and functional studies will be needed to further document the effect of this SNP upon Gγ-globin gene expression and to ascertain that this SNP represents a HPFH mutation. Hematological Data of Family Father Mother Son Son Age 52 44 13 9 Hb 15.7 14.1 13.2 13.8 MCV 82 77 75 75 Hb A2 2.5 % 3.0 % 3.4 % 3.3 % Hb F 10.2 % 0.7 % 0.7 % 5.9 %

scholarly journals Epigenetic Insights and Potential Modifiers as Therapeutic Targets in β–Thalassemia

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 755
Author(s):  
Nur Atikah Zakaria ◽  
Md Asiful Islam ◽  
Wan Zaidah Abdullah ◽  
Rosnah Bahar ◽  
Abdul Aziz Mohamed Yusoff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Presentation ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Dna Hypomethylation ◽  
Considerable Research ◽  
Non Coding Rna ◽  
Hbf Level ◽  
Long Non Coding Rna ◽  
Globin Gene Expression

Thalassemia, an inherited quantitative globin disorder, consists of two types, α– and β–thalassemia. β–thalassemia is a heterogeneous disease that can be asymptomatic, mild, or even severe. Considerable research has focused on investigating its underlying etiology. These studies found that DNA hypomethylation in the β–globin gene cluster is significantly related to fetal hemoglobin (HbF) elevation. Histone modification reactivates γ-globin gene expression in adults and increases β–globin expression. Down-regulation of γ–globin suppressor genes, i.e., BCL11A, KLF1, HBG-XMN1, HBS1L-MYB, and SOX6, elevates the HbF level. β–thalassemia severity is predictable through FLT1, ARG2, NOS2A, and MAP3K5 gene expression. NOS2A and MAP3K5 may predict the β–thalassemia patient’s response to hydroxyurea, a HbF-inducing drug. The transcription factors NRF2 and BACH1 work with antioxidant enzymes, i.e., PRDX1, PRDX2, TRX1, and SOD1, to protect erythrocytes from oxidative damage, thus increasing their lifespan. A single β–thalassemia-causing mutation can result in different phenotypes, and these are predictable by IGSF4 and LARP2 methylation as well as long non-coding RNA expression levels. Finally, the coinheritance of β–thalassemia with α–thalassemia ameliorates the β–thalassemia clinical presentation. In conclusion, the management of β–thalassemia is currently limited to genetic and epigenetic approaches, and numerous factors should be further explored in the future.

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 EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression

Blood ◽  
2015 ◽  
Vol 126 (16) ◽  
pp. 1930-1939 ◽  
Author(s):  
Aline Renneville ◽  
Peter Van Galen ◽  
Matthew C. Canver ◽  
Marie McConkey ◽  
John M. Krill-Burger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Locus ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cells ◽  
Adult Human ◽  
Key Points ◽  
Globin Gene Expression

Key Points EHMT1/2 inhibition increases human γ-globin and HbF expression, as well as mouse embryonic β-globin gene expression. EHMT1/2 inhibition decreases H3K9Me2 and increases H3K9Ac at the γ-globin gene locus in adult human erythroid cells.

An Erythoid-Specific Component of the Rhesus Stage Selector Protein, rNF-E4, Modulates γ-Globin Gene Expression Specifically.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1599-1599
Author(s):  
Ruiqiong Wu ◽  
Aurelie Desgardin ◽  
Stephen M. Jane ◽  
John M. Cunningham
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Globin Gene ◽  
Primate Species ◽  
Pcr Analysis ◽  
Gene Promoters ◽  
Fetal Stage ◽  
Globin Gene Expression ◽  
Selector Protein

Abstract Understanding the molecular mechanisms that regulate γ-globin gene expression is essential for development of new therapeutic strategies for individuals with sickle cell disease and β-thalassemia. We have previously identified a tissue- and developmentally- specific multiprotein transacting factor complex, the human stage selector protein (SSP), which facilitates the interaction of the g-globin gene promoters with the upstream locus control region enhancer in fetal erythoid cells. This complex interacts with the stage selector element (SSE) in the proximal g-globin promoter, a regulatory motif phylogenetically conserved in primate species with a distinct fetal stage of β-globin like gene expression. Given these observations, we hypothesized that a similar complex modulates γ-globin in the rhesus macaque, a non-human primate model that has been utilized to study β-globin like gene expression. We focused our efforts on NF-E4, given that a human isoform of this factor confers erythroid and fetal specificity to the SSP complex. Fetal liver erythroblasts were obtained from rhesus embryos and analyzed by reverse transcriptase(RT)-PCR analysis for NF-E4 expression. NF-E4 like transcripts were identified in day 60, 80 and 120 embryonic erythroblasts, but not other rhesus tissues, demonstrating an erythroid-specific pattern of expression. Utilizing 5′ RACE, we cloned a full length NF-E4 transcript, identifying an open reading frame encoding a 131 amino acid polypeptide. This 20kD polypeptide shares a high degree of homology with human NF-E4, especially in its carboxy-terminal domain. Like human NF-E4, GST pulldown chromatography confirmed the ability of the rhesus factor to interact directly with CP2 and ALY, the other core components of the SSP. To evaluate rNF-E4 function in vivo, we utilized retrovirally mediated gene transfer to enforce expression of this factor in K562 cells, a model of human fetal erythropoiesis. Initial co-immunoprecipitation studies confirmed the in vivo interaction of rNF-E4 with other components of the SSP. Interestingly, we observed a specific 3-fold induction of γ-globin gene expression in rNF-E4 expressing cells when compared to controls. Moreover, we demonstrated that, like enforced expression of human NF-E4, rNF-E4 induced a significant increase in ε-globin gene expression. Taken together, our results suggest a conservation of NF-E4 expression and function in species with a fetal stage of globin gene expression. Moreover, the identification of rNF-E4 provides a platform for the pre-clinical development of therapeutic agents that induce high levels of NF-E4 in adult erythroblasts.

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