Four Promoters of IRF5 Respond Distinctly to Stimuli and are Affected by Autoimmune-Risk Polymorphisms

Abstract

Introduction: Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis affect millions of people worldwide. Interferon regulatory factor 5 (IRF5) contains polymorphisms associated with these autoimmune diseases. Two of these functional polymorphisms are found upstream of the IRF5 gene. rs2004640, which is a single nucleotide polymorphism and the CGGGG insertion/deletion (indel) were studied. IRF5 uses four different promoters for its four first exons: 1A, 1B, 1C, and 1D. Each promoter was analyzed, including functional differences due to the autoimmune-risk polymorphisms.Results: IRF5 promoters were analyzed using ChIP-Seq data (ENCODE database) and the FactorBook database to define transcription factor binding sites. To verify promoter activity, the promoters were cloned into luciferase plasmids. Each construct exhibited luciferase activity. Exons 1A and 1D contain putative PU.1 and NFkB binding sites. Imiquimod, a Toll-like receptor 7 (TLR7) ligand, was used to activate these transcription factors. IRF5 levels were doubled after imiquimod treatment (p < 0.001), with specific increases in the 1A promoter (2.2-fold, p = 0.03) and 1D promoter (2.8-fold, p = 0.03). A putative binding site for p53, which affects apoptosis, was found in the promoter for exon 1B. However, site-directed mutagenesis of the p53 site showed no effect in a reporter assay.Conclusion: The IRF5 exon 1B promoter has been characterized, and the responses of each IRF5 promoter to TLR7 stimulation have been determined. Changes in promoter activity and gene expression are likely due to specific and distinct transcription factors that bind to each promoter. Since high expression of IRF5 contributes to the development of autoimmune disease, understanding the source of increased IRF5 levels is key to understanding autoimmune etiology.

Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFN-α Production in Women

“…Interestingly, an ERα binding site has been identified using the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu) at position 128561334-128561609 on human chromosome 7 which is 16,385bp upstream the IRF5 gene ( IRF5 chr7:128577994-128590088), suggesting that IRF5 may also be regulated by sex hormones in humans. Furthermore, one of the described IRF5 polymorphism in humans, the CGGGG indel, is associated with increased expression of IRF5 itself due to the presence of an additional SP1 binding site, an ERα cofactor (47, 69). Here, we found a significant correlation between IRF5 and Esr1 mRNA levels in pDCs from females but not in pDCs from males in humans, highlighting the dependency of IRF5 mRNA on estrogen signaling in females.…”

“…In addition, IRF5 polymorphisms have been associated with multiple autoimmune diseases, and in particular systemic lupus erythematous and rheumatoid arthritis (39–42), two autoimmune diseases characterized by over-production of type I IFN and by significant sex differences in prevalence. Autoimmune-risk haplotypes exhibit higher IRF5 levels (43) and are associated with increased levels of IFNα (44–46), suggesting that expression of IRF5 contributes to the development of autoimmune diseases (47). …”

Interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease

“…The fact that Irf5 expression is induced in human PBMC-derived or murine bone marrow-derived macrophages following differentiation with GM-CSF in vitro suggests transcriptional regulation by members of the STAT family of transcription factors (21)(22)(23). This is further highlighted in a study that used ChIP-seq datasets to identify transcription factor binding sites in the Irf5 locus, which identified a STAT2 binding site in the Exon 1C promoter, as well as numerous other binding sites across the 5' UTR including those for PU.1, IRF4, PAX5, TCF12, p53, EBF, AP1, Myc and NFκB (24). These transcription factors therefore also have the potential for regulation of Irf5 expression, although further experimental studies in multiple cell types are required to confirm this.…”

Endogenous retroviral promoter exaptation in human cancer

“…LOR1a LTRs are much less abundant compared to THE1 LTRs (Table 1) but are of similar age, with the IRF5 copy having inserted prior to New World-Old World primate divergence. IRF5 has multiple promoters/TSSs and complex transcription [118] and, contrary to the CSF1R case, the native promoters are not completely silent in HL. However, LTR activity correlates with strong overexpression of the IRF5 protein and transcript, above normal physiological levels [117].…”