Evidence that activation of the Egr-1 promoter by v-Raf involves serum response elements

The constitutively active serine/threonine kinase encoded by the v-raf oncogene, v-Raf, activates the Egr-1 promoter in transient expression assays. To characterize the v-Raf-responsive transcriptional control elements, deletion mutants of the Egr-1 promoter were used in transient expression assays. A v-Raf expression vector was co-transfected into NIH3T3 cells with reporter chloramphenicol acetyl transferase (CAT) expression vectors under the control of the Egr-1 promoter or the Egr-1 promoter containing various deletions. Responsiveness to v-Raf was restricted to a region that contained repeated CC(A/T)6GG sequences, known as CArG boxes. CArG boxes form the core of serum response elements (SREs). v-Raf-induced Egr-1 promoter activation was lost by removal of the four tandemly repeated SREs. This region, between -425 and -250, which was necessary for v-Raf responsiveness, was also found to be sufficient for maximal Egr-1 induction by v-Raf when placed upstream from a minimal heterologous promoter. Three out of four SREs from this region were able to respond to v-Raf, however the activation of the individual SREs was lower than the clustered SREs. This cluster of SREs has previously been shown to be responsive to several mitogenic stimuli and the oncogene v-src. Thus, the SREs contained in this cluster may be an important target for cell division signals.