Kang Hyo Jin, Kim Hee Jeong, Kim Sang Keun, Barouki Robert, Cho Chi-Heum, Khanna Kum Kum, Rosen Eliot M, Bae Insoo
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road NW, Washington, DC 20057, USA.
J Biol Chem. 2006 May 26;281(21):14654-62. doi: 10.1074/jbc.M601613200. Epub 2006 Mar 27.
Previously, we have reported that BRCA1 regulates the expression of various classes of genes, including genes involved in xenobiotic stress responses (Bae, I., Fan, S., Meng, Q., Rih, J. K., Kim, H. J., Kang, H. J., Xu, J., Goldberg, I. D., Jaiswal, A. K., and Rosen, E. M. (2004) Cancer Res. 64, 7893-7909). In the present study, we have investigated the effects of BRCA1 on xenobiotic stress-inducible gene expression. In response to aryl hydrocarbon receptor (AhR) ligands, cytoplasmic AhR becomes activated and then translocates to the nucleus where it forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT). Subsequently, the AhR.ARNT complex binds to the enhancer or promoter of genes containing a xenobiotic stress-responsive element and regulates the expression of multiple target genes including cytochrome P450 subfamily polypeptide 1 (CYP1A1). In this study, we have found that endogenous and overexpressed exogenous wild-type BRCA1 affect xenobiotic stress-induced CYP1A1 gene expression. Using a standard chromatin immunoprecipitation assay, we have demonstrated that BRCA1 is recruited to the promoter regions of CYP1A1 and CYP1B1 along with ARNT and/or AhR following xenobiotic exposure. Our findings suggest that BRCA1 may be physiologically important for mounting a normal response to xenobiotic insults and that it may function as a coactivator for ARNT activity. Using immunoprecipitation, Western blotting, and glutathione S-transferase capture assays, a xenobiotic-independent interaction between BRCA1 and ARNT has been identified, although it is not yet known whether this is a direct or indirect interaction. We have also found that the inducibility of CYP1A1 and CYP1B1 transcripts following xenobiotic stress was significantly attenuated in BRCA1 knockdown cells. This reduced inducibility is associated with an altered stability of ARNT and was almost completely reversed in cells transfected with an ARNT expression vector. Finally, we have found that xenobiotic (TCDD) treatments of breast cancer cells containing reduced levels of BRCA1 cause the transcription factor ARNT to become unstable.
此前，我们曾报道BRCA1可调节各类基因的表达，包括参与外源性应激反应的基因（裴，I.，范，S.，孟，Q.，里，J. K.，金，H. J.，康，H. J.，徐，J.，戈德堡，I. D.，贾斯瓦尔，A. K.，以及罗森，E. M.（2004年）《癌症研究》64卷，7893 - 7909页）。在本研究中，我们探究了BRCA1对外源性应激诱导基因表达的影响。响应芳烃受体（AhR）配体时，胞质AhR被激活，然后转位至细胞核，在细胞核中它与芳烃受体核转运体（ARNT）形成复合物。随后，AhR-ARNT复合物结合至含有外源性应激反应元件的基因的增强子或启动子，并调节包括细胞色素P450亚家族多肽1（CYP1A1）在内的多个靶基因的表达。在本研究中，我们发现内源性和过表达的外源性野生型BRCA1会影响外源性应激诱导的CYP1A1基因表达。使用标准的染色质免疫沉淀分析，我们证明在接触外源性物质后，BRCA1会与ARNT和/或AhR一起被募集至CYP1A1和CYP1B1的启动子区域。我们的研究结果表明，BRCA1对外源性损伤产生正常反应可能具有重要的生理意义，并且它可能作为ARNT活性的共激活因子发挥作用。通过免疫沉淀、蛋白质印迹和谷胱甘肽S-转移酶捕获分析，已鉴定出BRCA1与ARNT之间存在一种不依赖外源性物质的相互作用，尽管尚不清楚这是直接相互作用还是间接相互作用。我们还发现，在BRCA1敲低的细胞中，外源性应激后CYP1A1和CYP1B1转录本的诱导性显著减弱。这种诱导性降低与ARNT稳定性的改变有关，并且在用ARNT表达载体转染的细胞中几乎完全逆转。最后，我们发现，用外源性物质（TCDD）处理BRCA1水平降低的乳腺癌细胞会导致转录因子ARNT变得不稳定。
