Regulation Mechanism Of Human TIM-1 Gene Expression And Association Study Of TIM-1 Gene Polymorphisms And Asthma In A Chinese Han Population

Asthma is a chronic inflammaroy pulmonary disorder caused by mix reaction of different inflammatory lymphocytes, such as eosinophile granulocyte, labrocyte and T cells. The main symptoms include the obstruction of bronchial tubes, the increase of airway responsiveness, the airway inflammation and remodelling. Asthma is thought to arise from an imbalance in T helper (Th1-Th2) type 1 immune regulation,resulting in the driving of the development of Th2-biased immune responses and the overproduction of cytokines such as interleukin-4 (IL-4), IL-5, IL-9 and IL-13, which mediate allergic inflammation. Although environmental factors play important roles in the development of asthma, there is a strong genetic predisposition in asthma patients. Several asthma susceptibility genes have been identified through whole genome scan and candidate gene approaches. TIM-1, a member of the T cell immunoglobulin and mucin domain (TIM) gene family, is one such gene that figures prominently in such investigation.The TIM gene family is located on human chromosome 5q31-33, a region linked to asthma. It contains 3 members in human beings (TIM-1, TIM-3 and TIM-4), all of which have recently been implicated in the control of T cell-mediated immune responses.TIM-1 is selectively expressed on activated CD4+ T cells and its expression was sustained preferentially on Th2 cells. The TIM-1 gene contains many polymorphisms, of which exon 4 variations have been intensively studied for evaluating the role in pathogenesis of allergic diseases. Privious association studies in a Chinese Han population, show that a functional SNP in TIM-1 gene regulatory region is associated with asthma. This SNP may affect TIM-1 gene expression. To better understand whether or how TIM-1 gene plays a role in the pathogenesis of asthma, we characterized the promoter of human TIM-1 gene and genotyped five additonal SNPs of TIM-1 gene in 569 asthma patients and 578 healthy controls.PART ONE:Regulation Mechanism of Human TIM-1 Gene ExpressionAlthough much attention has been focused on the association of TIM-1 gene polymorphisms with asthma, the promoter elements and transacting factors directly involved in human TIM-1 gene transcription have not been identified.As the first step to characterize the promoter of human TIM-1 gene, we performed a computer-based searching for the putative transcription factor binding sites within about 2Kb fragment upstream the transcription start site of human TIM-1 gene using MatInspector software. We failed to find any canonical TATA box or CAAT box. However, several potential transcription factor binding motifs were identified in the promoter region. This result was used as the reference for the design of luciferase reporter vectors.In order to localize the core promoter sequence, we generated a series of progressive 5'deletion constructs. Each construct and pRL-TK vector were cotransfected into Chang Liver and 293T cells, and then the relative luciferase activity was determined. And then, three potential binding motifs were suggested, located at-98bp to -28bp,-28bp to -9bp and +422bp to +458 bp, respectively. Similar tendency was observed in two different cell lines tested.To characterzie the binding motif between -98 to -28, computer-based analysis was conducted and a putative E2F4 binding site was identified. To determine whether this E2F4 is critical for constitutive promoter activity of human TIM-1 gene, point mutations were introduced into TIM1-61 construct which changed the E2F4 consensus core sequence from GATCGCGCCA to GATCTTTTCA and generated E2F4 mutant construct (TIM1- E2F4-Mut). The luciferase activity driven by mutant E2F4 construct (TIM1- E2F4-Mut) was reduced to a range as observed in the construct that lacks E2F4 binding site(TIM1-28), indicating that the E2F4 binding site between -98 and -28 of the human TIM-1 promoter contributes to the basal activity of the human TIM-1 transcription;To examine whether the putative Spl binding site located between -28 to -9 mediates the transcriptional activity of TIM-1 promoter, point mutations were introduced into TIM1-28 construct which changed Sp1 consensus core sequence from AGGCGG toATTTTG and generated Sp1 mutant construct (TIM1-Sp1-Mut). The luciferase activity driven by mutant Sp1 construct (TIM1-Sp1-Mut) was reduced to a range as observed in the construct that lacks Sp1 binding site(TIM1-9), indicating that the Spl binding site between -27 and -8 of the human TIM-1 promoter contributes to the basal activity of the human TIM-1 transcription;To examine whether the putative GFI1 binding site located between +422 to +458 mediates the transcriptional activity of TIM-1 promoter, point mutations were introduced into TIM1-+445 construct which changed GFI consensus core sequence from ATT to CCC and generated GFI1 mutant construct (TIM1- GFI1-Mut). The luciferase activity driven by mutant GFI1 construct (TIM1- GFI1-Mut) was reduced to a range as observed in the construct that lacks GFI1 binding site(TIM1-+458), indicating that the GFI1 binding site between +422 to +458bp of the human TIM-1 promoter contributes to the basal activity of the human TIM-1 transcription;To further confirm the essential role of E2F4,Sp1 and GFI1 binding motifs, we conducted EMSA to examine the binding of endogenous E2F4,Sp1 and GFI1 to each binding site. The retarded mobility of the oligonucleotides was observed in the presence of nuclear extracts. The DNA-protein complex was significantly competed by 25- and 100-fold molar excess of unlabeled wild-type oligonucleotides, but not by the same amount of mutant E2F4,Sp1 and GFI1 oligonucleotides. These results indicate that the E2F4,Spl and GFI1 element in this region specifically binds with endogenous protein.Taken together, our deletion and mutation analysis as well as EMSA demonstrated that E2F4 binding site between -98 to -28,Sp1 binding site located between -28 to -9 and GFI binding site located between +422 to +458 are responsible for the constitutive transcription.PART TWO:Association study of TIM-1 gene polymorphisms and asthma in a Chinese Han populationAsthma is thought to arise from an imbalance in Thl-Th2 immune regulation. Therefore, the genes involved in the regulation of CD4+T cell differentiation might be the asthma susceptibility genes. TIM-1 is one of the recently identified genes implicated in the regulation of Thl and Th2 immune responses, and was implicated as an asthma susceptibility gene in previous studies. In order to further evaluate the effects of the TIM-1 gene polymorphisms on asthma susceptibility in the Chinese Han population, we genotyped five additional SNPs, rs4235719, rsl553318, rs953568, rs1039438 and rs6878732, in 569 asthma patients and 578 healthy controls. SNP rs4235719 is located in the upstream regulatory region of the gene, rs1553318 and rs953568 are in intron 4; rs1039438 and rs6878732 are tag SNPs based on the HapMap data, each representing a different linkage disequilibrium block.Each SNP was genotyped by Polymerase Chain Reaction-Fragment Length Polymorphism (PCR-RFLP) or Primer Induced Restriction Analysis -PCR (PIRA-PCR). The distribution of five SNPs were in agreement with Hardy-Weinberg equilibrium in both patients and controls (P values all greater than the 0.05). The genotype and allele frequencies of each SNP were compared between the patients and normal controls, and no significant difference was observed in our Chinese cohort, indicating that these five SNPs were not associated with asthma suscepbility in the Chinese Han population.Previously we gentoyped five SNPs and found one of them was associated with asthma. To further confirm these results, we genotyped these five SNPs in additional samples. Consistent with the previous data, rs41297579, rs6149307, rs45439103 and rs34670839 were not associated with asthma in total samples, whereas rs9313422 was associated with asthma.We analyzed the linkage disequilibrium (LD) among the 10 SNPs. No strong LD situation was detected between them. Haplotype analyses identified three common haplotypes. The hap 3 was associated with increased asthma susceptibitlity in our population (odds ratio 2.001,95% confidence interval 1.492-2.686). TIM-1 is a recently identified asthma susceptibility gene and was verified in our previous studies. In order to further evaluate the effects of the TIM-1 gene polymorphisms on asthma susceptibility in the Chinese Han population, we genotyped five SNPs in addition to those tested in our previous study, rs4235719, rsl553318, rs953568, rs1039438 and rs6878732, in 569 asthma patients and 578 healthy controls. Each SNP was genotyped by PCR-RFLP or Primer Induced Restriction Analysis -PCR (PIRA-PCR). The genotype and allele frequencies of each SNP were compared between the patients and normal controls, and no significant difference was observed in our Chinese cohort. Combined with the previous data of rs41297579, rs9313422, rs6149307, rs45439103 and rs34670839, we analyzed the haplotype of the 10 SNPs and identified three common haplotypes. The h ap 3 was associated with increased asthma susceptibility in our population (odds ratio 2.001, 95% confidence interval 1.492-2.686).