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 inducible by genotoxic treatments. Regulation of transcriptional activation of rhoB is still unclear. Here we show that cells lacking either p53 or c-Fos 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) necessary for stimulation of rhoB by UV light. By means of immunoprecipitation we found that an ATF-2-specific antibody co-precipitates the (32)P-labeled 0.17 kb rhoB fragment, whereas an anti-AP1 antibody did not. Since no consensus sequence for binding of ATF-2 is present within the rhoB promoter, ATF-2 is likely to be associated with another factor that binds to the minimal promoter. Deletion analysis and site-directed mutagenesis of the 0.17 kb rhoB fragment revealed a CCAAT box to be an essential requirement for stimulation of rhoB by UV light and methyl methanesulfonate. Moreover, immunoprecipitation experiments showed that the CCAAT-binding factor NF-YA is complexed with ATF-2. Overall, the data strongly indicate that transcriptional activation 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.
Fritz
G
G
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. fritz@mail.uni-mainz.de
Kaina
B
B
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000528:radiation effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000528:radiation effects*; D014466:Ultraviolet Rays; D020743:rhoB GTP-Binding Protein / Q000235:genetics*
D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; C492060:Nfya protein, mouse; D009841:Oligonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D020743:rhoB GTP-Binding Protein