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.

编码Ras相关GTP酶RhoB（Ras-related GTPase RhoB）的基因可被基因毒性处理诱导为即刻早期基因。目前对于rhoB转录激活的调控机制仍不明确。本研究发现，缺失p53或c-Fos的细胞在UV照射后，rhoB的诱导水平与野生型细胞无显著差异，表明这些转录因子对于rhoB mRNA表达的激活并非必需。对UV照射及未照射细胞的提取物进行检测，发现其与一段长0.17 kb的rhoB启动子片段具有相似的DNA结合活性，该片段包含UV诱导rhoB表达所需的功能元件。通过免疫沉淀实验，我们发现ATF-2特异性抗体可共沉淀经32P标记的0.17 kb rhoB启动子片段，而抗AP1抗体则无此效果。由于rhoB启动子区域内不存在ATF-2的共有结合序列，因此ATF-2很可能通过与另一结合于核心启动子的因子形成复合物发挥作用。对0.17 kb的rhoB片段进行缺失分析及定点突变实验表明，CCAAT盒是UV照射及甲磺酸甲酯（methyl methanesulfonate）诱导rhoB表达的必需元件。此外，免疫沉淀实验证实CCAAT结合因子NF-YA可与ATF-2形成复合物。综上，本研究数据充分表明，基因毒性应激介导的rhoB基因转录激活依赖于CCAAT盒，且CCAAT结合因子与ATF-2的相互作用可触发rhoB的应激诱导表达。