POS0362 INVESTIGATING THE ANKYLOSING SPONDYLITIS-ASSOCIATED REGULATORY SNPS AT THE RUNX3 LOCUS WITH A FUNCTIONAL GENOMICS APPROACH

Abstract

Background:Of the >100 genetic associations with ankylosing spondylitis (AS), RUNX3 transcription factor (TF) involved in diverse immunological processes, is robustly (10−15) associated.1 The biggest challenge is to understand the mechanism behind this association. We demonstrated the association between AS and the single nucleotide polymorphism (SNP) rs4648889 located in a 2kb regulatory locus upstream of the RUNX3 promoter can be explained by allele-specific effects on TF recruitment that alter gene expression, specifically in CD8+ T-cells.2 We recently dissected the full plethora of TFs affected by rs4648889 AS-associated allele showing the NuRD complex and IRF5 differentially bound and having a crucial role in CD8+ T-cells function3. Nevertheless, other post-GWAS approaches are needed to elucidate the function of RUNX3 AS-associated SNPs (i.e. the lead SNP rs6600247).Objectives:The purpose of this work is to define the functional effect of rs6622047 in CD8+ T-cells and monocytes. In specific, we expect to define the impact of AS-associated allele to TF binding and to evaluate chromosome looping between rs6600247 and the RUNX3 promoter.Methods:The epigenetic landscape of SNP rs6600247 was defined using Roadmap database. In vitro functional studies were performed to characterize the effects of this SNP on TFs binding. Chromosome conformation capture (3C) provided critical functional evidence for looping among AS-associated SNPs and the RUNX3 promoter.Results:(1) In silico data revealed a c-MYC ChIP-seq peak in GM12878 lymphoblastoid cells overlapping rs6600247; (2) Mobility shift assays (EMSAs) and WB-EMSAs showed reduced DNA/protein binding in the presence of the AS-risk allele in CD14+ monocytes. C-MYC binding-site is disrupted and binding abolished in the presence of the AS-risk allele; (3) 3C experiments indicate low interaction frequency between SNP rs6600247 and RUNX3 promoter.Conclusion:The enhancer upstream the RUNX3 gene has a plausible functional role in AS, probably by regulating gene transcription and DNA looping. These observations are critically important in defining dysregulated pathways and potential therapeutic drug targets.