A negative regulatory element within the proximal promoter region of the rat ornithine decarboxylase gene.
A putative Ets site with a core of GGAA located at nt -88 to -85 of the rat ornithine decarboxylase (ODC) gene was characterized by site-directed mutagenesis and transient expression assays. Mutation of this site, when in pODClux2m, which contains a cluster of four Sp1-binding sites, resulted in a 2.6-fold increase in basal promoter activity in untreated cells, whereas the ratio of activity in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells relative to the ratio in untreated cells (the induction ratio) remained largely unchanged. However, when the mutation was in pODClux168, which contains only a single Sp1-binding site (GC box V), it caused little alteration to either basal promoter activity or TPA induction ratio. A protein of 55-60 kDa was found specifically bound to this site, as shown by ultraviolet cross-linking assay. In competition assay and methylation interference assay, this protein was shown to occupy the GGAA core, although it showed no antigenic relation to c-Ets-1 in an supershift assay. We suggest that this protein binds specifically to the GGAA core and functions to inhibit activation of the ODC promoter by distal elements, including the upstream Sp1 sites.
Zhao
B
B
The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA.
Kumar
A P
AP
Butler
A P
AP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D019175:DNA Methylation; D015870:Gene Expression / Q000187:drug effects; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D008156:Luciferases; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate