van der Watt Pauline J, Leaner Virna D
Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Biochim Biophys Acta. 2011 Jul;1809(7):316-26. doi: 10.1016/j.bbagrm.2011.05.017. Epub 2011 Jun 13.
The nuclear exporter protein, Crm1, plays a key role in normal cell functioning, mediating the nucleo-cytoplasmic transport of cargo proteins. Elevated Crm1 expression has recently been identified in various tumours; however, the mechanisms driving its expression have not been investigated to date. In this study we identified the Crm1 promoter and factors associated with its elevated expression and with its repression under conditions of DNA damage. The -1405 to +99 Crm1 promoter region was found to be significantly more active in cancer and transformed cells compared to normal, and the -175 to +99 region identified as responsible for the differential activity. Mutation of two CCAAT boxes and a GC box within this region significantly diminished Crm1 promoter activity and ChIP analysis revealed binding of NFY and Sp1 to these sites, with increased binding in transformed and cancer cells. In addition, p53 was found to repress Crm1 promoter activity, after induction with doxorubicin, with p53 siRNA blocking the effect. Crm1 promoter constructs with mutated CCAAT boxes were significantly less responsive to p53 repression, and in vivo binding of NFY to the CCAAT boxes was diminished upon p53 binding, suggesting that p53 mediates repression of the Crm1 promoter via interfering with NFY. This was confirmed using NFY knock-down cells, in which Crm1 promoter activity was significantly less responsive to p53. In vitro EMSAs revealed that NFY and p53 bind the CCAAT boxes as a single complex under conditions of DNA damage. In summary, this study is a first to analyse Crm1 promoter regulation and reveals NFY and Sp1 as contributors to Crm1 overexpression in cancer. In addition, this study reveals that Crm1 transcription is inhibited by DNA damage and that the mechanism of inhibition involves p53 interfering with NFY function.
核输出蛋白Crm1在正常细胞功能中发挥关键作用，介导货物蛋白的核质运输。最近在各种肿瘤中发现Crm1表达升高；然而，驱动其表达的机制迄今尚未得到研究。在本研究中，我们确定了Crm1启动子以及与其表达升高和在DNA损伤条件下受抑制相关的因子。发现-1405至+99的Crm1启动子区域在癌症和转化细胞中比正常细胞具有显著更高的活性，并且-175至+99区域被确定为负责这种差异活性。该区域内两个CCAAT框和一个GC框的突变显著降低了Crm1启动子活性，染色质免疫沉淀分析显示NFY和Sp1与这些位点结合，在转化细胞和癌细胞中的结合增加。此外，在用阿霉素诱导后，发现p53可抑制Crm1启动子活性，p53小干扰RNA可阻断该效应。具有突变CCAAT框的Crm1启动子构建体对p53抑制的反应明显降低，并且p53结合后NFY与CCAAT框的体内结合减少，表明p53通过干扰NFY介导Crm1启动子的抑制。使用NFY敲低细胞证实了这一点，其中Crm1启动子活性对p53的反应明显降低。体外电泳迁移率变动分析表明，在DNA损伤条件下，NFY和p53作为单一复合物结合CCAAT框。总之，本研究首次分析了Crm1启动子调控，并揭示NFY和Sp1是癌症中Crm1过表达的促成因素。此外，本研究还揭示Crm1转录受到DNA损伤的抑制，并且抑制机制涉及p53干扰NFY功能。
