Isolation of a genomic clone containing the promoter region of the human ATP binding cassette (ABC) transporter, ABCB6.
We previously reported on the isolation of a new rat ATP binding cassette (ABC) transporter, ABCB6. We now report the isolation of the full-length cDNA and genomic clones containing the human ABCB6 gene. ABCB6 is 100% identical to the cloned MTABC3 human ABC transporter and contains the typical ABC signature, Walker A and B motifs. We found that HuABCB6 is expressed at low levels in normal human liver. We found that ABCB6 was overexpressed in human hepatocellular carcinomas compared to paired surrounding non-malignant tissue. We found that there was no difference in ABCB6 gene copy between human liver cancer and its paired non-malignant tissue. Because HuABCB6 was overexpressed in human cancers compared to peri-tumoral tissue in the absence of gene amplification, transcriptional regulation may play an important role in its expression. Therefore, we isolated a 14 kb genomic DNA clone containing the HuABCB6 promoter and 5'-flanking region. The 5'-flanking region contains a CpG island, lacks an appropriately positioned TATA element and contains a number of putative transcription factor binding sites. Two transcription start sites were identified by S1 nuclease mapping at -274 and -296 bp from the start codon. Transient transfection of the HuABCB6 promoter constructs (HuABCB6/1.68, 1.39, 1.13, 0.90, 0.52) containing the luciferase reporter gene resulted in a 1100-2300-fold increase in luciferase activity compared to the empty vector control whereas HuABCB6/1.68 subcloned in the reverse orientation resulted in no activity. We observed a significant decrease in luciferase activity with the promoter constructs, HuABCB6/0.25, 0.15 and 0.06, which indicates that an orientation-dependent functional promoter is contained within our previously predicted promoter region of -315 bp to -565 bp as deletion of this 250 bp sequence resulted in a loss of promoter activity.
Emadi-Konjin
H Pasha
HP
Division of Gastroenterology and Nutrition, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, ON, Canada.
Zhang
Hui
H
Anandan
Vasuki
V
Sun
Daxi
D
Schuetz
John
J
Furuya
Katryn N
KN
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014162:Transfection; D014407:Tumor Cells, Cultured
C107991:ABCB6 protein, human; D018528:ATP-Binding Cassette Transporters; C491540:Abcb6 protein, rat; D018076:DNA, Complementary; D012333:RNA, Messenger; D015719:Single-Strand Specific DNA and RNA Endonucleases