Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene.
NF-R2 is a DNA-binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF-R2 binds within the promoter's -126 and -102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF-R2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S-300, DEAE-Sepharose, heparin-Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF-R2 runs as a single protein of 75 kDa on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF-R2 binding. These results suggest that NF-R2 may be involved in the positive regulation of the MDR1 gene transcription.
Takatori
T
T
Institute of Applied Microbiology, University of Tokyo.
Ogura
M
M
Tsuruo
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004351:Drug Resistance / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D015534:Trans-Activators; D015500:Chloramphenicol O-Acetyltransferase