肿瘤抑制基因XAF1启动子序列中活性IRF-1作用元件的鉴定

Identification of a high affinity IRF-1 binding element in XAF1 promoter of tumor suppressing gene

目的在XAF1的转录起始序列中鉴定一具有高度活性的IRF-1作用元件。方法通过生物信息学分析发现XAF1转录起始处(-30^-38nt)有一潜在的干扰素(IFN)调节因子1结合元件(IRF-E),与同义IRF-E序列同源性达76.2%,命名为IRFE-XAF1。应用凝胶电泳迁移实验(EMSA)检测IRFE-XAF1的核蛋白结合活性,并分别定点突变其核心序列内-34nt和序列旁-28nt,检测突变后XAF1启动子活性变化及对IFN-α作用的影响。结果EMSA实验发现32P标记的含IRFE-XAF1的双链寡核苷酸DNA探针可与细胞核蛋白结合,且可被IRF-E同义探针阻断,定点突变后结合活性丧失,证实该IRFE-XAF1位点具有与转录因子结合的活性。含该IRFE-XAF1位点的XAF1启动子序列具有启动子活性且可被IFN-α诱导,IRFE-XAF1序列定点突变后启动子活性明显降低,其中-34nt突变后完全消除了IFN-α上调启动子活性的作用。结论XAF1的转录起始序列具有一高度活性的IRF-E,其序列为-38ntGAAACGAAA-30nt,这一发现提示XAF1是IFN-α诱导肿瘤细胞分化的作用基因。

Objective To identify a high affinity IRF-1 binding element in the translational start site of XAF1 promoter. Methods By the bioinformatics analysis, a putative IFN regulatory factor 1 binding element （IRF-E）, named as IRFE-XAF1, was identified from -30nt to -38nt of the XAF1 gene, with 76.2% homogeneity with the synonymous IRF-E sequence. Electrophoresis mobility shift assay （EMSA） was performed to confirm the binding capacity of IRFE-XAF1. Two site-directed mutations were made, one mutation site was outside of the IRF-E region （-28nt） and another located at the center of IRF-E （-34nt）. The promoter of XAF1 after mutation was examined, including its binding activity and response to IFN-α. Results It was found by EMSA assay that the doublestranded oligonucleotide DNA probe, containing IRFE-XAF1 and labeled with 32P, may be connected to the nuclear protein, and blocked by the unlabeled synonymous IRF-1 probe （cold probe）. The binding capacity of IRF-E was lost after site-directed mutation. The XAF1 promoter containing IRFE-XAF1 site had the priming activity, and could be induced by IFN-α. The priming activity declined markedly after site-directed mutation of IRFE-XAF1, and -34 site mutation completely eliminated the effect of IFN-α. Conclusion A high affinity of IRF-E is found in -30nt to -38nt region upstream of ATG initiator codon of XAF1 gene. The code sequence is -38nt-GAAACGAAA--30nt. The present study suggests that XAF1 is one of the genes with which IFN-α may induce the differentiation of cancer cells.