Purification and characterization of NF-R1 that regulates the expression of the human multidrug resistance (MDR1) gene.
We have purified a protein, NF-R1, that specifically binds to two unrelated motifs--the ATTCAGTCA motif and the GC-box motif--on the MDR1 proximal promoter. Purified NF-R1 has been confirmed by southwestern blotting to be a 110-kDa protein. Methylation interference analysis revealed that the nucleotides were in close contact with purified NF-R1 on the ATTCAGTCA and GC-box motifs. The nucleotides were required for the binding of NF-R1, as seen by competition gel mobility shift assay using point mutated oligonucleotides. In a CAT expression assay using the corresponding point-mutated MDR1 promoter fused to a CAT gene, binding inhibition of NF-R1 to the promoter resulted in 2- to 3-fold increases of CAT activity, as compared to the intact promoter in Adriamycin-resistant K562 cells. Thus NF-R1 has a relation to the negative regulation of the MDR1 gene transcription.
Institute of Applied Microbiology, University of Tokyo, Japan.
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M
Ogura
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T
Takatori
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Tsuruo
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D004247:DNA