Identification of a high affinity IRF-1 binding element in XAF1 promoter of tumor suppressing gene / 解放军医学杂志

ABSTRACT

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-?. 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-?. The priming activity declined markedly after site-directed mutation of IRFE-XAF1, and -34 site mutation completely eliminated the effect of IFN-?. 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-? may induce the differentiation of cancer cells.