Functional characterization of the transcription silencer element located within the human Pi class glutathione...

We have previously demonstrated enhanced transcriptional activity of the human Pi class glutathione S-transferase (GSTP1) promoter in a multidrug-resistant derivative (VCREMS) of the human mammary carcinoma cell line, MCF7 (Moffat, G. J., McLaren, A. W., and Wolf, C. R. (1994) J. Biol. Chem. 269, 16397-16402). Furthermore, we have identified an essential sequence (C1; -70 to -59) within the GSTP1 promoter that bound a Jun-Fos heterodimer in VCREMS but not in MCF7 cells. These present studies have examined the negative regulatory element (-105 to -86), which acted to suppress GSTP1 transcription in MCF7 cells. Mutational analysis of this silencer element further defined the repressor binding site to be located between nucleotides -97 and -90. In vitro DNA binding assays suggested that the repressor exerted its action by causing displacement of the essential non-AP-1-like MCF7 C1 complex. However, the addition of MCF7 nuclear extract did not disrupt binding of the VCREMS Jun-Fos C1 complex to the GSTP1 promoter. Furthermore, upstream insertion of the GSTP1 silencer element failed to inhibit activity of a heterologous promoter in MCF7 cells. These results highlighted the cell and promoter specificity of the GSTP1 transcriptional repressor and implicated a functional requirement for contact between the repressor and C1 complex. In this regard, the introduction of half-helical turns between the silencer and the C1 element abrogated repressor activity, thus leading to the hypothesis that a direct interaction between the repressor and C1 complex was required to suppress GSTP1 transcription. Moreover, these findings suggest that cell-specific differences in the composition of the C1 nuclear complex may dictate repressor activity.