Transcriptional activation of the small GTPase gene rhoB by genotoxic stress is regulated via a CCAAT element

The gene encoding the Ras-related GTPase RhoB specific is immediate-early i nducible by genotoxic treatments. Regulation of transcriptional activation of rhoB is still unclear. Here we show that cells lacking either p53 or c-F os are not different from wild-type cells with respect to the level of rhoB induction upon UV irradiation, indicating that these transcription factors are not crucial for stimulation of rhoB mRNA expression. Extracts from UV- irradiated and non-irradiated cells revealed similar DNA-binding activities to a 0.17 kb rhoB promoter fragment harboring the functional element(s) ne cessary for stimulation of rhoB by UV light. By means of immunoprecipitatio n we found that an ATF-2-specific antibody co-precipitates the P-32-labeled 0.17 kb rhoB fragment, whereas an anti-AP1 antibody did not. Since no cons ensus sequence for binding of ATF-P is present within the rhoB promoter, AT F-2 is likely to be associated with another factor that binds to the minima l promoter. Deletion analysis and site-directed mutagenesis of the 0.17 kb rhoB fragment revealed a CCAAT box to be an essential requirement for stimu lation of rhoB by UV light and methyl methanesulfonate. Moreover, immunopre cipitation experiments showed that the CCAAT-binding factor HE-YA is comple xed with ATF-P. Overall, the data strongly indicate that transcriptional ac tivation of the rhoB gene by genotoxic stress is regulated via a CCAAT box and that interaction of CCAAT-binding factor and ATF-2 triggers the stress- inducible expression of rhoB.