A RUNX3 enhancer polymorphism associated with ankylosing spondylitis influences recruitment of Interferon Regulatory Factor 5 and factors of the Nucleosome Remodelling Deacetylase Complex in CD8+ T-cells
Vecellio M., Cortes A., Bonham S., Selmi C., Knight JC., Fischer R., Brown MA., Wordsworth BP., Cohen CJ.
<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Objectives</jats:title><jats:p>To investigate the functional consequences of the single nucleotide polymorphism <jats:italic>rs4648889</jats:italic> in a putative enhancer upstream of the <jats:italic>RUNX3</jats:italic> promoter strongly associated with ankylosing spondylitis (AS).</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>The effects of <jats:italic>rs4648889</jats:italic> on transcription factor (TF) binding were tested by DNA pull-down and quantitative mass spectrometry. The results were validated by electrophoretic mobility gel shift assays (EMSA), Western blot (WB) analysis of the pulled-down eluates, and chromatin immuno-precipitation (ChIP)-qPCR.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Several TFs showed differential allelic binding to a 50bp DNA probe spanning <jats:italic>rs4648889</jats:italic>. Binding was increased to the AS-risk A allele for IKZF3 (<jats:italic>aiolos</jats:italic>) in nuclear extracts from CD8+ T-cells (3.7-fold, p<0.03) and several components of the NUcleosome Remodeling Deacetylase (NuRD) complex, including Chromodomain-Helicase-DNA-binding protein 4 (3.6-fold, p<0.05) and Retinoblastoma-Binding Protein 4 (4.1-fold, p<0.02). In contrast, binding of interferon regulatory factor (IRF) 5 was increased to the AS-protective G allele. These results were confirmed by EMSA, WB and ChIP-qPCR.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The association of AS with <jats:italic>rs4648889</jats:italic> most likely results from its influence on the binding of this enhancer-like region to TFs, including IRF5, IKZF3 and members of the NuRD complex. Further investigation of these factors and RUNX3-related pathways may reveal important new therapeutic possibilities in AS.</jats:p></jats:sec>