Effect Of ERCC4 Promoter Haplotypes On Transcription Activity

Lung cancer is one of the most common cancers worldwide,which has the highest mortality rate among all the cancers.Tobacco caused DNA damage or lesion plays an important role in lung cancer genesis.Polycyclic aromatic hydrocarbons, aromatic amines,and nitrosamines involved in the tobacco smoke have been implicated as the major mutagenic carcinogens responsible for DNA adduct formation. Accumulated DNA adducts lead to an enhanced probability for DNA damage,which could impede or block translation and replication,followed by potential mutation and cancer ultimately.Nucleotide excision repair(NER),one of the most versatile DNA repair systems,is the primary means by which smoking-induced genomic mangle can be removed.So,impairment of this DNA repair system may arouse susceptibility to some characteristic human disorders,such as lung cancer.Human ERCC4/XPF(Excision repair cross-complementing group 4)locates in 16p13.3-13.13 and it encodes one of the key enzymes in NER pathway.This enzyme forms a complex with protein ERCC1 and the complex can not only recognize damaged DNA but also specifically excise the 5' end of the damaged DNA fragment. ERCC4 gene deficits may lead to genetic instability and carcinogenesis.A recent study showed that in a Chinese population(n=1010),the-644C/T(rs3136038) polymorphism in the ERCC4 promoter was significantly associated with the risk of lung cancer.To elucidate the underlying molecular mechanism of the association,we investigated the relationship between the polymorphisms in the ERCC4 promoter and ERCC4 mRNA transcription.We predicted the transcription factors that bind the polymorphisms sites by bioinformatics.We further validated this result by EMSAs, and analyzed whether SNPs of-644C/T and -357A/C(rs6498486)on the transcriptional regulation affect binding of transcription factors.The luciferase report system experiment indicated that the transcription activity of haplotypes in the promoter of ERCC4 has significant difference.A promoter fragment containing major alleles(-644C/-357A)showed the highest activity in HELF cells, compared with other haplotypes.Real-time quantitative PCR showed that cigarette smoke extract(CSE) significantly inhibited ERCC4 gene expression.Transfection experiments in the presence of CSE indicated that the major haplotype of-644C/-357A has the best protective effect for CSE treatment.Electrophoretic mobility shift assays(EMSA)revealed that CREB and CRE-BP1 possibly were the transcription factors that bind the region containing positions -644 and -357.The major haplotype of -644C/-357A of in ERCC4 promoter could significantly enhanced binding ability with CREB and CRE-BP1.Relation between the -644C/T(rs3136038)genotypes and ERCC4 expression level showed that the minor allele homozygote TT carriers had lower expression level of ERCC4 gene than the major allele homozygote CC.Our results suggested that genetic and environmental factors may interact to regulate ERCC4 expression.We postulated that the transcription regulation of ERCC4 gene might abide by the following pattern:chemical carcinogens→stress signals including DNA damage→activation of cellular signal wansduction pathway→change of gene expression→enhanced or decreased DNA repair ability.The ERCC4 with different genotypes might have different expression levels to external conditions of DNA damage.Our research may contribute to elucidating the etiology of lung cancer.