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.