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 muta genesis and transient expression assays. Mutation of this site, when in pOD Clux2m, which contains a cluster of four Spl-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 ce lls relative to the ratio in untreated cells (the induction ratio) remained largely unchanged. However, when the mutation was in pODClux168, which con tains only a single Spl-binding site (GC box V), it caused little alteratio n 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 crosslinking assay. In competition assay and methylation interference assay , this protein was shown to occupy the GGAA core, although it showed no ant igenic relation to c-Ets-l in an supershift assay. We suggest that this pro tein binds specifically to the GGAA core and functions to inhibit activatio n of the ODC promoter by distal elements, including the upstream Spl sites. (C) 2000 Wiley-Liss, Inc.