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Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization
Published on: October 6, 2019
IRF5:RelA interaction targets inflammatory genes in macrophages
David G Saliba1, Andreas Heger2, Hayley L Eames1
1Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Oxford OX37FY, UK.
Abstract:
Interferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities.
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