DNA­-binding of IRF3, IRF5 and IRF7 transcription factors

The Interferon Regulatory Factors (IRFs) are essential regulators of the innate immune response to viruses. IRFs 3, 5, and 7 drive type­-1­-interferon and cytokine production in a cell-­type and stimulus­-specific manner, suggesting regulatory complexity. IRFs ­3, ­5 and ­7 bind DNA as phosphorylation-­activated dimers and are classically described as binding a doublet of the canonical IRF binding site: 5'-­AANNGAAA­-3’. IRF­3/5/7 have both common and distinct gene targets and the simple canonical motif does not capture the regulatory complexity of IRF­-dependent gene expression. Previous studies, both low and high throughput, have refined our understanding of IRF regulation, yet a systematic comparison of DNA binding preferences for active, dimeric IRF complexes has not been performed. To address this, we designed an IRF­-specific, custom protein-­binding microarray (PBM) that includes synthetic IRF binding sites as well as type-1-interferon and cytokine promoters. Using constitutively active, phosphomimetic IRF­3, 5 and 7 dimers, we find key DNA binding differences for these factors with implications for IRF-dependent gene regulation. Five protein binding microarray (PBM) experiments of human Interferon Regulatory Factor (IRF) transcription factors were performed. These PBM experiments involved binding GST-tagged, constitutively active IRF3, IRF5, IRF7 as well as the DNA binding domain mutants IRF5-K96S and IRF7-S101K to double-stranded 180K Agilent microarrays. DNA sequences tested on the array include: 1) predicted IRF binding sites from human and mouse type-I-interferon proximal promoters; 2) synthetic IRF binding sites with every possible single base pair substitution. All genomic and synthetic binding sites are centered within the same constant flanking sequence. Each DNA sequence is present between 4 and 6 times as replicate array spots: synthetic IRF binding sites have 6 replicate spots, genomic IRF binding sites have 5 replicate spots, and background control sequences have 4 replicate spots. Here we report the normalized signal intensity for each DNA spot.