| Ankylosing spondylitis (AS) is a common inflammatory arthritis which attaches the axial skeleton and peripheral joints and is influenced by multiple factors including genetics, immunology and environment. Systemic lupus erythematosus (SLE) is characteristic of multisystem disorder and it is also considered as a classic complex and heterogeneous autoimmune disease as well as AS. Although there are many genetic studies reporting susceptible genes associated with SLE by Genome-wide association study (GWAS) in Chinese population rather than AS, both of them are characterized by excessive activation of the immune system. Increasing evidence has suggested that many of the known SLE-associated loci overlapped with those of other immune-related diseases. Consequently, the susceptible gene of SLE in East Asian population and rheumatoid arthritis (RA) in Caucasian population, ETS1, may be associated with AS as well.ETS1 is a member of the ETS family of transcription factors affecting transcriptional activity by binding to the GGAA/T motif in target genes, which is encoded by ETS1 gene that was initially discovered as the pro-oncogene corresponding to v-ets of the avian erythroblastosis virus (E26). As an important transcription factor, ETS1 is widely expressed in various tumor cells, lymphoid tissues, vascular endothelium and lacteal glandular epithelium, and ETS1 regulates numerous crucial biological processes both in normal cells and tumors. In Etsl deficient mice, there are several aberrations in B cell differentiation noted consisting of alteration about the number of IgM- and IgG- secreting cells in the peripheral lymphoid organs and bone marrow and prevention of TLR-induced plasma cell terminal differentiation probably attribute to the regulation of expression of Blimp1 and Pax5. Etsl deficient mice have variant defects in T cells lineage, for instance, a failure to maintain the normal ratio of differentiated Thl and Th2 cells through controlling specific cytokines, impairment in negative regulation of Th17 cells depending on IL-2 and deficiency in giving rise to normal number of Treg cells due to dysregulation in expressing Foxp3.Previously established evidence has confirmed that ETS1 was a critical factor in biological process, cellular component and molecular function. By use of animal experiments, ETS1 was pointed to perform a vital role in the development and differentiation of immune cells through various pathways in both innate and adaptive immune response.Accordingly, we investigated the association of ETS1 and AS in terms of clinical and immunological overlap between AS and other autoimmune diseases. If there is any difference in the expression of ETS1 between patients and controls, the reason to explain this phenomenon will be identified in our study. Further study concerning the target gene, who might be participate into the mechanism of AS and SLE, regulated by ETS1 will also be discovered to guide the therapy of preventing autoimmunity in clinic. The main results acquired in this study are displayed as follow:Part IAssociation studies of ETS1 with AS and SLEIn order to investigate whether variants in ETS1 confer susceptibility to AS in Han Chinese, The genotyping involving the TaqMan method was implemented in 1,015 patients with AS and 1,132 sex-, age-matched healthy controls from a health check-up center of Shandong Provincial Hospital and Qilu Hospital who have been long living in Shandong Province, and also in the replication samples of 352 AS patients and sex-, age-matched 400 healthy controls who were long-term residents in Ningxia, a northwest region in China. All patients had no history of inflammatory bowel disease and/or psoriasis and met the modified New York criteria for AS. In the meantime,398 patients with SLE and 480 sex-, age-matched healthy controls all from a Chinese Han population living in Shandong were recruited to participate in the case-control study about the susceptibility to SLE.No novel variant was found in all 9 exons and 1000bp of the upstream sequence by sequencing of DNA samples from 100 AS individuals. Then we selected 7 SNPs (rs12574073, rs1128334, rs4937333, rs4254089, rs7951925, rs7116578 and rs4937342) in ETS1 with minor allele frequency≥0.05 and r2>0.8 in data for Han Chinese in Beijing (HCB) in HapMap (www.hapmap.org) and genotyped by TaqMan SNP genotyping assay using assay-on demand probes. Frequency of the rs1128334 allele A was significantly higher in AS patients (odds ratio [OR]=1.204,95% confidence interval [95%CI] 1.058-1.371, P=0.005) than in healthy controls. Even after Bonferroni correction, the association remained significant (Pc=0.035).rs12574073 and rs4937333 were associated with AS but not after Bonferroni correction. Then, we replicated the associated SNP rs1128334 in the Ningxia cohort of 352 cases and 400 matched controls. The minor allele frequency of rs1128334 was greater for cases than controls (OR=1.305, 95% CI 1.055-1.615, P=0.015). rs1128334 was in strong LD with rs12574073 and rs4937333. As expected, the haplotype results were similar to the single-site association results, and haplotype TAT formed by rs12574073,rs1128334 and rs4937333 was strongly associated with increased risk of AS (OR=1.24,95% CI 1.05-1.47, P=0.01). A statistically significant difference was observed in the frequency of minor allele of all 3 SNPs between cases and controls (rs12574073:OR=1.593,95% CI 1.290-1.968, P<0.001;rs1128334:OR=1.832,95%CI 1.489-2.256, P<0.001;rs4937333:OR=1.515, 95%CI 1.240-1.850, P<0.001)We have demonstrated that ETS1 was associated with AS, thus adding to the list of loci showing overlap between AS, RA and SLE. Besides, we verified the association of rsl 128334 and AS in 2 populations, Han Chinese from Shandong and Ningxia, which enhanced the credibility of our findings. The replication study regarding the association between genetic polymorphisms of ETS1 and the risk of SLE in northern region population in Chinese Han population manifested that rsl2574073, rs1 128334 and rs4937333 might contribute to susceptibility to SLE in Han Chinese people.Part IIIdentifying the functional variant in ETS1 gene associated with autoimmune diseasesAs mentioned above, ETS1 was associated with AS, thus adding to the list of loci showing overlap between AS and SLE, and we also identified an SNP, rs1 128334, located in the 3’UTR of ETS1, which was significantly associated with AS in Han Chinese people. Whether it was likely to be the causal variant of the two antoimmune diseases susceptibility remained unclear then. According to the general study strategy about functional variants, we examined the mRNA levels of ETS1 in patients and healthy controls at first. Our study measured ETS1 mRNA levels in PBMCs derived from AS or SLE patients and respective controls. We observed that ETS1 mRNA expression in patients was significantly lower than in normal controls (P<0.0001). To determine whether rsl 128334 was associated with ETS1 expression, we genotyped 161 healthy controls for rs1128334 to compare the ETS1 expression status in subjects with different genotypes of the SNP. Our finding revealed carriers with the risk allele A of rsl 128334 exhibited significantly lower ETS1 mRNA expression than those with the allele G (P=0.0024). Although ETS13’UTR sequence bearing the non-risk allele of rs1128334 matches a predicted binding site of miR-300, different alleles of this variant was not found to affect the binding affinity of miR-300 and further influence the expression of the target gene.Imputation study carried out by The University of Hong Kong and present studies proved rs6590330 have stronger contribution than any other variant including rs1128334 to association. As expected, rs1128334 is in almost absolutely LD (r2=0.97) with rs6590330, another variant that is located in the downstream region of ETS1. rs6590330 was decided to be investigated as the candidate causal variant instead of rs1128334 on account of their same genotyping results in the same individuals.The intergenic SNP might affect gene expression by involving in transcription regulation, therefore the relative dual luciferase activity was used to represent the transcriptional activity of allele A or G on rs6590330. As expected, the activity of allele A was lower than it was of allele G in HEK293 and HeLa cells, especially in HeLa cells (P=0.0265). Furthermore, different alleles were also cotransfected with genomic DNA fragments derived from promoter of ETS1 (-842bp upstream from the transcriptional start site) in order to prove the different effect carried out by allele A or G, respectively. The activity test result was consistent with the aforementioned observation in HeLa cells (.P=0.0006). Hence, these data indicated that the allele A is associated with the decreasing expression of ETS1.Is the transcription of ETS1 modulated by transcription factor binding affinity to different alleles? On the basis of the prediction in MatInspector and PROMOE analysis software, electrophoretic mobility shift assay (EMSA), supershift and chromatin immunoprecipitation (ChIP) were employed to demonstrate Spl could specifically bind to rs6590330 site in vitro and in vivo. Moreover, this transcription factor performed higher affinity interacting with allele G than allele A, which might explain the underlying molecular mechanism of the correlation between allele A of rs6590330 and reduced expression of ETS1.For purpose of pinpointing the effect of Spl to different alleles of rs6590330, luciferase activity analysis and expression vector of Spl were applied to indicate Spl might transactivate ETS1 by binding to allele G more than to allele A.The results in this part implied that transcription factor Spl could bind to rs6590330 with high affinity to allele G, which probably gave rise to the association between allele A of rs6590330 and reduced expression of ETS1.Part Ⅲ Preliminary identification of target genes regulated by ETS1 by ChIP-seqAlthough substantial research has confirmed ETS1 could directly participate into a variety of immune response pathways and the transcription of T cell-specific cytokines, the mechanism of it effecting immunity in B cells is undiscovered. In order to investigate the potential transcriptional regulation of ETS1 involving immune response, Chromatin immunoprecipitation (ChIP) was performed in Raji cells, a kind of human B cell lymphoma expressing ETS1 constitutively, to get the genomic DNA fragments binding with ETS1 antibody specifically in vivo.Positive fragments were then sequenced to construct the ChIP library and were analyzed to harvest the high quality reads so as to complete genome mapping by MACS software. As expected, the distribution trend of the potential transcriptional sites on chromosomes was in accord with the volume of them and most of the sites were located in chrl to chr7. According to the relative position of peaks to the known genes, the peak distribution in different genomic features was 13.37% in upstream (defined as TSS-20kb-TSS-2kb),6.44% in promoter (defined as TSS-2kb-TSS+2kb),32.64% in intron (defined as USCS RefSeq introns), only 1.64% in exon(defined as USCS RefSeq exons) and 45.92% in intergenic region (defined as the other genomic regions not included in the 4 categories above), which indicated ETS1 could not only regulate the basal promoter, but also participate in the expression of target genes by binding with their further regions. The peak-associated genes were used to conduct GO analysis to functionally annotate these enriched regions. As described in the GO results, the target genes of ETS1 could distribute in many areas covering biological process, cellular component and molecular function. The peak-associated genes were also used to conduct KEGG Pathway analysis to infer the pathways that these peak-associated genes involved in. TRAF5, CREB3L4, CXCL5, TAB2, CREB5 and CREBL2 were 6 candidates involved in TNF signaling pathway, which suggested ETS1 might partially participate in the process of the death of lymphocytes through TNF signaling pathway.The candidate target genes distributed in promoter region were analyzed to verify the accuracy of sequencing by ChIP in Raji cells. The promoter of CDK9, YY1, IL-17RA, SOX4, CXCR5, PTPN12 and GMPR2 were specifically recognized by ETS1 as the bioinformatics analysis predicted.In this part of study, we explored the target genes of ETS1 in human lymphocytes to understand the function of this transcription factor in the process of autoimmune diseases. Consequently, ETS1 might affect various pathways in immune response by regulating relative target genes to prevent individuals from autoimmune diseases. Analysis of target genes of ETS 1 would provide a novel direction to target therapy for autoimmune diseases in clinic. |
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