Kim Bo-Kyung, Im Joo-Young, Han Gyoonhee, Lee Woo-Jung, Won Kyoung-Jae, Chung Kyung-Sook, Lee Kyeong, Ban Hyun Seung, Song KyungBin, Won Misun
BioMedical Genomics Research Center, KRIBB, Daejeon 305-806, Korea.
Dept. of Biotechnology, Yonsei University, Seoul 129-749, Korea.
Biochim Biophys Acta. 2014 May;1839(5):364-73. doi: 10.1016/j.bbagrm.2014.03.004. Epub 2014 Mar 15.
The anti-cancer agent NSC126188 induces apoptosis of stomach carcinoma NUGC-3 cells by inducing RhoB expression. Here, we present that the p300 binding site in the RhoB promoter is crucial for the binding of p300 and its partner transcription factors to activate RhoB transcription in NSC126188-mediated apoptosis. NSC126188 increased expression of p300 and c-Jun. Conversely, knockdown of p300 decreased RhoB expression in the presence of NSC126188. We found that poly(ADP-ribose) polymerase-1 (PARP-1) was associated with the p300 binding site and that PARP-1 knockdown inhibited NSC126188-mediated RhoB expression. In the cells treated with NSC126188, p300, PARP-1, and c-Jun interacted and bound the p300 binding site. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed strong p300 binding and weak c-Jun binding at the p300 binding site of RhoB promoter in cells treated with NSC126188. We also demonstrated that c-Jun played a crucial role in p300 binding. However, PARP-1 did not directly bind the p300 binding site, suggesting a bridging role between p300 and c-Jun. Electrophoretic mobility shift assays demonstrated a complex comprising p300/c-Jun/PARP-1 that bound wild type, but not a mutated, p300 binding site. In addition, overexpression of p300, PARP-1, or c-Jun dramatically enhanced RhoB promoter activity when it contained the wild type sequence but not mutated sequences, indicating the crucial role of the p300 binding site in NSC126188-induced transcription of RhoB. Taken together, these data suggest that p300 is recruited and cooperates with c-Jun and PARP-1 at the p300 binding site to activate RhoB transcription during NSC126188-mediated apoptosis.
抗癌药物NSC126188通过诱导RhoB表达诱导胃癌NUGC - 3细胞凋亡。在此，我们提出RhoB启动子中的p300结合位点对于p300及其伙伴转录因子在NSC126188介导的凋亡中结合以激活RhoB转录至关重要。NSC126188增加了p300和c-Jun的表达。相反，在存在NSC126188的情况下，p300的敲低降低了RhoB的表达。我们发现聚（ADP-核糖）聚合酶-1（PARP-1）与p300结合位点相关，并且PARP-1的敲低抑制了NSC126188介导的RhoB表达。在用NSC126188处理的细胞中，p300、PARP-1和c-Jun相互作用并结合p300结合位点。此外，染色质免疫沉淀（ChIP）分析显示在用NSC126188处理的细胞中，RhoB启动子的p300结合位点处有强p300结合和弱c-Jun结合。我们还证明c-Jun在p300结合中起关键作用。然而，PARP-1不直接结合p300结合位点，表明其在p300和c-Jun之间起桥梁作用。电泳迁移率变动分析表明存在一个由p300/c-Jun/PARP-1组成的复合物，该复合物结合野生型而非突变型p300结合位点。此外，当p300、PARP-1或c-Jun过表达时，若RhoB启动子包含野生型序列而非突变序列，则会显著增强RhoB启动子活性，表明p300结合位点在NSC126188诱导的RhoB转录中起关键作用。综上所述，这些数据表明在NSC126188介导的凋亡过程中，p300在p300结合位点被募集并与c-Jun和PARP-1协同作用以激活RhoB转录。
