title: Functional characterization of the transcription silencer element located within the human Pi class glutathione S-transferase promoter.
abstract: 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.
authors.affiliation: Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, DD1 9SY, Scotland, United Kingdom.
authors.forename: G J
authors.initials: GJ
authors.lastname: Moffat
authors.forename: A W
authors.initials: AW
authors.lastname: McLaren
authors.forename: C R
authors.initials: CR
authors.lastname: Wolf
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005260:Female; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase