Novel Regulatory Regions in the Interleukin 6 Gene and Implications for Disease/Gene Association Studies
1JM Samuel, 2AJP Smith, 2SE Humphries, 1P Woo
1Department Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London, United Kingdom, 2Centre for Cardiovascular Genetics, University of London, University Street, London, United Kingdom
Interleukin-6 (IL-6, encoded by gene IL6) is an important pleiotropic cytokine that is regulated at the transcriptional level. To date, most work on its regulation has focused on a 1.2kb region 5' from the start of transcription. We have identified the -174 G allele of the IL6 gene as a susceptibility loci in systemic JIA . This variant was also shown to influence IL6 transcription and serum levels. However, our recent investigation of the functionality of -174 G/C, with additional polymorphisms in the IL6 promoter, indicated a more complex regulatory haplotype extending further upstream of the previously analysed promoter region. The involvement of a much larger upstream region in the regulation of IL6 is described in this report. Comparative genomics analysis has identified a 120kb region which contains blocks of sequence conservation between human and rodent genomes. Additionally, the 15kb region proximal to the start of transcription contains ten highly homologous sequence blocks of between 100-250bp. By means of a reporter gene assay, a novel 105bp transcriptionally active region, located 5kb upstream from the start of transcription, was identified. Further evidence that this sequence is involved in the regulation of IL6 is indicated by the binding of protein(s) to this region using electrophoretic mobility shift assays. To determine if any common variation was present in this region it was sequenced using the DNA from 24 healthy individuals. No variation in sequence was identified. Both the -174 C and G alleles have been associated with numerous autoimmune and inflammatory diseases. These associations have been inconsistent between studies, so it is possible that these differences can be explained by the presence of additional nucleotide variation in other regions important for transcriptional regulation. Our study has identified a region important in the regulation of IL6 transcription, but no common polymorphisms were identified in this region. Work is currently focusing on identifying polymorphisms, within the 120kb conserved upstream region of IL6, that could explain the anomalies in the associations between IL6 -174 genotype and IL-6 levels. In summary, the results of this study suggest that the regulation of IL6 expression involves a much larger upstream region than previously examined, and the control of IL6 transcription is likely to be regulated by a complex mechanism of modular cis-regulatory elements.