FUNCTIONAL-CHARACTERIZATION OF THE TRANSCRIPTION SILENCER ELEMENT LOCATED WITHIN THE HUMAN PI-CLASS GLUTATHIONE-S-TRANSFERASE PROMOTER

We have previously demonstrated enhanced transcriptional activity of t he human Pi class glutathione S-transferase (GSTP1) promoter in a mult idrug-resistant derivative (VCREMS) of the human mammary carcinoma cel l line, MCF7 (Moffat, G, J,, McLaren, A. W., and Wolf, C. R (1994) J. Biol. Chem. 269, 16397-16402), Furthermore, we have identified an esse ntial sequence (C1; -70 to -59) within the GSTP1 promoter that bound a Jun-Fos heterodimer in VCREMS but not in MCF7 cells, These present st udies have examined the negative regulatory element (-105 to -86), whi ch acted to suppress GSTP1 transcription in MCF7 cells. Mutational ana lysis of this silencer element further defined the repressor binding s ite to be located between nucleotides -97 and -90, In vitro DNA bindin g assays suggested that the repressor exerted its action by causing di splacement of the essential non-AP-1-like MCF7 C1 complex, However, th e addition of MCF7 nuclear extract did not disrupt binding of the VCRE MS jun-Fos C1 complex to the GSTP1 promoter, Furthermore, upstream ins ertion of the GSTP1 silencer element failed to inhibit activity of a h eterologous promoter in MCF7 cells, These results highlighted the cell and promoter specificity of the GSTP1 transcriptional repressor and i mplicated a functional requirement for contact between the repressor a nd 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 r epressor 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.