Distinct TATA motifs regulate differential expression of human metallothionein I genes MT-IF and MT-IG.
In this report, we have measured the cadmium (Cd2+)-induced expression of all known metallothionein I (MT-I) mRNAs in a human hepatoma cell line, Hep G2. Among the human MT-I gene family promoters, marked sequence conservation exists; despite this, the mRNA accumulation level for each species was found to be quite unique. This differential Cd2+ induction of MT-I family members provides an ideal opportunity to assess whether the characteristic response results from subtle isoform-specific variations in promoter structure. Accordingly, we have examined the mechanism for differential expression of two isoforms, MT-IG and MT-IF, by transient transfection into Hep G2 cells. In the presence of Cd2+, MT-IG promoter activity and endogenous mRNA level were, respectively, 4.7- and 3-fold greater than those of MT-IF. This close correlation between promoter activity and mRNA accumulation strongly suggests that differential expression occurs at the level of transcription. The difference in Cd(2+)-stimulated activity was found to be conferred by 240- and 243-base pair promoter fragments spanning nucleotides -174 to +66 and -172 to +71 of the MT-IG and MT-IF genes, respectively. One of the most striking nonhomologies between the promoters is a single A (TATAAA) to C (TATCAA) transversion in the TATA motifs of MT-IG and MT-IF genes, respectively. To determine whether such a subtle change in the TATA motif could account for the marked differences in promoter function, we constructed MT-IG-TATCA and MT-IF-TATAA promoters and measured their activities in transient transfection and cell-free transcription assays. Results of both assays showed a profound difference between the two motifs that paralleled the difference in Cd(2+)-stimulated MT-IG and MT-IF mRNA levels. In summary, we have shown that differential regulation of two MT-I promoters is primarily due to a single base alteration in their TATA motifs.
Shworak
N W
NW
Department of Biological Sciences, University of Calgary, Alberta, Canada.
O'Connor
T
T
Wong
N C
NC
Gedamu
L
L
D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D004247:DNA; D017931:DNA Primers; D005786:Gene Expression Regulation; D006801:Humans; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D002104:Cadmium; D004247:DNA; D008668:Metallothionein