In vivo footprinting using N-ethyl,N-nitrosourea: improved resolution of the DNA-protein interactions in the human gamma-globin gene promoter region.
N-Ethyl, N-nitrosourea (ENU) was used as a probing agent in conjunction with a modified ligation-mediated polymerase chain reaction in a new in vivo footprinting procedure. In the present work, we examined the promoter region of the human gamma-globin gene under both uninduced and hemin-induced conditions in K562 cells. In the course of comparing this method with the standard dimethyl sulfate (DMS) in vivo method and previously reported results, we were able to verify our new method. However, discrepancies between these methods were observed at the stage selector element, -50 region, of gamma-globin promoter. Our in vivo footprinting result showed DNA-protein interaction at this region under the hemin-induced condition which was not revealed by the conventional DMS in vivo footprinting method. This approach, using ENU-modified in vivo footprinting, is now being applied to clarify the mechanism of cytotoxic drug-induced fetal hemoglobin augmentation.
Kim
I H
IH
Laboratory of Chemical Biology, National Institutes of Health, Bethesda, Maryland 20892, USA.
Rodgers
G P
GP
D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005038:Ethylnitrosourea / Q000378:metabolism*; D005319:Fetal Hemoglobin / Q000378:metabolism; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011506:Proteins; D013463:Sulfuric Acid Esters; D006427:Hemin; D005914:Globins; D004247:DNA; D005319:Fetal Hemoglobin; C007482:dimethyl sulfate; D005038:Ethylnitrosourea