Evidence that SP1 modulates transcriptional activity of the multidrug resistance-associated protein gene.
In previous studies, we have cloned and sequenced a 5'-end region of the multidrug resistance-associated protein (MRP) gene that contains promoter activity as assessed through transient transfections of constructs contained in a pCAT basic reporter plasmid. In the present study, using a series of deletion mutants, evidence was obtained that the SP1 binding sites contained in the promoter are essential for optimal MRP transcriptional activity. These results were supported by the finding that introduction of site-specific mutations into the wild-type SP1 sequence produced a major reduction in CAT activity. DNase I protection assays also demonstrated that SP1 sites are protected from hydrolysis with proteins from nuclei of a variety of cell lines. Gel mobility-shift assays with proteins extracted from CHO, HeLa, HL60, or HL60/ADR demonstrated the presence of a protein that bound to the wild-type SP1 sequence but not to an SP1 sequence containing site-specific mutations. The mobility shift with nuclear extracts was closely similar to that occurring after incubating purified SP1 protein with wild-type SP1 sequence. DNA supershift experiments with antibody to SP1 strongly suggest that the complexes formed with nuclear extracts contain the SP1 protein.
Zhu
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Division of Biology, Kansas State University, Manhattan 66506, USA.
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D016466:CHO Cells / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D018983:DNA Footprinting; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D004317:Doxorubicin; D015500:Chloramphenicol O-Acetyltransferase