title: A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells.
abstract: We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factor beta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 +/- 1.0%; EPO + TGF-B = 10.8 +/- 1.9%; EPO + SCF = 19.1 +/- 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 +/- 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 +/- 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 +/- 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation.
authors.affiliation: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Natarajan V
authors.initials: NV
authors.lastname: Bhanu
authors.forename: Tiffany A
authors.initials: TA
authors.lastname: Trice
authors.forename: Y Terry
authors.initials: YT
authors.lastname: Lee
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Gantt
authors.forename: Patricia
authors.initials: P
authors.lastname: Oneal
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Schwartz
authors.forename: Pierre
authors.initials: P
authors.lastname: Noel
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Miller
mesh_terms: D000328:Adult; D002256:Carbonic Anhydrases / Q000096:biosynthesis / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology; D005312:Fetal Blood / Q000378:metabolism; D020868:Gene Silencing; D006454:Hemoglobins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051899:Smad2 Protein; D019089:Stem Cell Factor / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D005719:gamma-Globulins / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D006454:Hemoglobins; D012333:RNA, Messenger; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D019089:Stem Cell Factor; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D005719:gamma-Globulins; D004921:Erythropoietin; D020929:Mitogen-Activated Protein Kinase Kinases; D002256:Carbonic Anhydrases