Regulation of transcription of the human MRP7 gene. Characteristics of the basal promoter and identification of tumor-derived transcripts encoding additional 5' end heterogeneity.
Studies focusing on the transcriptional regulation of MRP7 (multidrug resistance associated protein 7) gene expression in human tumor cells are described. As shown by real-time RT-PCR, expression of the MRP7 gene compared to the expression of the MRP1, 2 and 3 genes was less variable among the different cell types. MRP1, 2, 3 and 7 gene expression was highest in HepG2 cells compared to expression in CWR22Rv1 and TSU-PR1 cells. MRP7 gene expression was less than expression of the MRP1 and 2 genes in HepG2 cells but similar to MRP3 gene expression in this cell type and similar to or greater than expression of the MRP1, 2 and 3 genes in CWR22Rv1 and TSU-PR1 cells. Functional deletion analysis, in situ mutagenesis and electromobility shift assays (EMSA) showed that basal MRP7 promoter activity relied upon a proximal segment of the 5' flanking region 169 to 257 nt in length bearing an E2F site acting cooperatively with two closely positioned Sp1 sites. Two additional Sp1 sites further downstream were of secondary importance. The sequence of the E2F site was noncanonical and its interaction with E2F protein was confirmed by a competitive EMSA using a consensus E2F oligonucleotide probe and by demonstrating a supershift with the antibody against the E2F4 and E2F5 pocket protein, p107. 5' RACE carried out with CWR22Rv1 and HepG2 cells detected a single transcription start site (tsp) distal to the basal promoter and identified two new MRP7 transcripts with very short 5' UTR sequences compared to transcripts found by others in nontumorous human tissue. This 5' end heterogeneity infers a more complex intron-exon composition than hitherto shown.
Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA.
Magdalena
M
Dabrowska
Francis M
FM
Sirotnak
D024506:5' Flanking Region / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D020121:5' Untranslated Regions; D008156:Luciferases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C421142:ABCC10 protein, human; C498885:Abcc10 protein, mouse; C421109:multidrug resistance-associated protein 1