The functional genetics of respiratory syncytial virus induced bronchiolitis : an investigation into the transcriptional regulation of IL8 and LTA

Hacking, Doug (2004). The functional genetics of respiratory syncytial virus induced bronchiolitis : an investigation into the transcriptional regulation of IL8 and LTA. PhD thesis The Open University.



This thesis explores the extent to which natural variation in DNA sequence may influence the transcriptional regulation of genes that play a critical role in the immune response to infection. As a model system we began by examining Interleukin-8 (IL-8) as it has been implicated in the pathogenesis of a very common infection namely, RSV-induced bronchiolitis. Previously, we have described an association between bronchiolitis disease severity and a specific IL8 haplotype comprising six single nucleotide polymorphisms (SNPs) (-251A/+396G/+781T/+1238deLA./+1633T/+2767T, haplotype 2). Here we investigated the functional basis for this association by measuring haplotype-spedfic transcription in vivo in human primary cells. We found a significant increase in transcript level derived from the IL-8 haplotype 2 relative to the mirror haplotype 1 (-251T/+396T/+781C/+1238insA/+1633C/+2767A) in respiratory epithelial cells but not in lymphocytes. A promoter polymorphism, -251 A, present on the high producer haplotype, had no clear and significant effect on the allele-spedfic level of transcription when analyzed in reporter gene experiments in human respiratory epithelial cell lines. We proceeded to systematically screen for allele-spedfic protein-DNA binding in this functional haplotype which revealed significant differential binding at the +781T/C polymorphism. C/EBP β was identified as being part of a transcription factor binding complex which preferentially bound in the presence of +781T allele. These results suggest that the mechanism for disease susceptibility to RSV-induced bronchiolitis may occur through a haplotype-spedfic increase in IL8 transcription which may be mediated by functional polymorphisms within that haplotype.
However, the unusual mirror haplotype structure of IL8 in European populations gave little indication of which SNP within the haplotype was likely to be functional in vivo. In order to demonstrate the principle that a non-coding polymorphism may alter transcriptional regulation in vivo I used a second technique, namely the haplotype-specific chromatin immunopredpitation (haploChIP), which once optimised could be applied to the IL8 locus. In order to optimise haploChIP I studied a second model system namely the tumor necrosis factor/lymphotoxin-a TNF/LTA) locus and used it to measures the differences in the loading of RNA polymerase between alleles in EBV-immortalized B lymphocytes. Consistent with previous studies I demonstrated that allele-spedfic RNA polymerase loading was independently assodated with the LTA+80, LTA+368 and LTA+723 polymorphisms. This association was refined to demonstrate that the polymerase loading at this locus appeared to be determined in vivo by the LTA+80A and LTA+368C alleles both of which occur on the same haplotype. These data imply that HaploChIP could be utilised in understanding the transcriptional regulation of IL8. Furthermore it shows that functional SNPs may act in concert to influence transcription. This may mean that in future the functional basis for allele-spedfic effects at critical candidate genes wül have to be dissected in vivo at the level of the haplotype.

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