DISTINCT TATA MOTIFS REGULATE DIFFERENTIAL EXPRESSION OF HUMAN METALLOTHIONEIN-I GENES MT-I(F) AND MT-I(G)

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 seque nce conservation exists; despite this, the mRNA accumulation level for each species was found to be quite unique. This differential Cd2+ ind uction of MT-I family members provides an ideal opportunity to assess whether the characteristic response results from subtle isoform-specif ic variations in promoter structure. Accordingly, we have examined the mechanism for differential expression of two isoforms, MT-I(G) and MT -I(F), by transient transfection into Hep G2 cells. In the presence of Cd2+, MT-I(G) promoter activity and endogenous mRNA level were, respe ctively, 4.7- and 3-fold greater than those of MT-I(F). This close cor relation between promoter activity and mRNA accumulation strongly sugg ests that differential expression occurs at the level of transcription . The difference in Cd2+-stimulated activity was found to be conferred by 240- and 243-base pair promoter fragments spanning nucleotides -17 4 to +66 and -172 to +71 of the MT-I(G) and MT-I(F) genes, respectivel y. One of the most striking nonhomologies between the promoters is a s ingle A (TATAAA) to C (TATCAA) transversion in the TATA motifs of MT-I (G) and MT-I(F) genes, respectively. To determine whether such a subtl e change in the TATA motif could account for the marked differences in promoter function, we constructed MT-I(G)-TATCA and MT-I(F)-TATAA pro moters and measured their activities in transient transfection and cel l-free transcription assays. Results of both assays showed a profound difference between the two motifs that paralleled the difference in Cd 2+-stimulated MT-I(G) and MT-I(F) mRNA levels. In summary, we have sho wn that differential regulation of two MT-I promoters is primarily due to a single base alteration in their TATA motifs.