Alterations in protein-DNA interactions in the gamma-globin gene promoter in response to butyrate therapy.
The mechanisms by which pharmacologic agents stimulate gamma-globin gene expression in beta-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with beta-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of gamma-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in gamma-globin protein synthetic levels above baseline gamma globin ratios and a relative decrease in beta-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the gamma-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, alphaCP2, 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 gamma-globin gene expression is being altered strongly suggest that nuclear protein binding, including alphaCP2, to the BRE-G1 region of the gamma-globin gene promoter mediates butyrate activity on gamma-globin gene expression.
Hemoglobinopathy-Thalassemia Research Unit and Cancer Research Center, Departments of Pharmacology and Experimental Therapeutics, Pediatrics, and Medicine, Boston University School of Medicine, Boston, MA, USA.
T
T
Ikuta
Y W
YW
Kan
P S
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Swerdlow
D V
DV
Faller
S P
SP
Perrine
D000293:Adolescent; D000328:Adult; D000755:Anemia, Sickle Cell / Q000097:blood / Q000188:drug therapy / Q000235:genetics; D001483:Base Sequence; D002087:Butyrates / Q000494:pharmacology* / Q000627:therapeutic use; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004900:Erythroblasts / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D005914:Globins / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D017086:beta-Thalassemia / Q000097:blood / Q000188:drug therapy* / Q000235:genetics
D002087:Butyrates; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D005914:Globins; D004247:DNA; D005319:Fetal Hemoglobin