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.

Keywords: IRF5; alternative promoters; autoimmune disease risk; interferon; systemic lupus erythematosus.

Figure 1

Interferon regulatory factor 5 mRNA and the position of the rs2004640 SNP and CGGGG indel. (A) rs2004640 is at the splice acceptor site for exon 1B, and the CGGGG indel is 64 bp upstream from the transcription start site for exon 1A. The genomic region of IRF5 is drawn to scale, but with introns reduced in size 10:1. The protein coding and untranslated regions are shown above. Only one first exon is used per RNA transcript; therefore each first exon corresponds to the untranslated region for that transcript. (B) The position of the rs2004640 SNP on pre-mRNA. Before splicing, the messenger RNA has either a U (encoded by the risk T allele) or G. The colored letters shown in the WebLogo (the nucleotide stacks of varying heights represent the consensus recognition sites for the spliceosome. The height of the stack represents how often those nucleotides are found at that position, and thus the high GT represents a strong preference for recognizing GT at the intron boundary. This matches in the risk T allele (GT at the intron boundary), but not the protective allele (GG at the intron boundary). A person homozygous for the protective allele cannot splice IRF5 mRNA that begins with exon 1B. Instead of a functional protein, the resultant mRNA would encode a non-functional protein and be targeted for non-sense mediated decay. Splice junction WebLogos are from Stephens and Schneider (7). (C) The CGGGG indel is an insertion/deletion of a CGGGG repeat upstream of exon 1A, and it is part of exon 1A’s promoter. When there are four copies, additional SP1 transcription factors (which bind to GGCGG) can bind to the promoter, altering transcription levels. UTR, untranslated region; SNP, single nucleotide polymorphism.

Figure 2

Promoter analysis of each first exon of IRF5. (A) ENCODE data shows results of ChIP-Seq analyses in the promoter region of IRF5. (B) The consensus search terms generated from FactorBook, with the TCF12 consensus binding site as an example (35). This data was used to manually define the nucleotide search terms shown. (C) The final analysis of potential binding sites is shown along the genomic DNA promoter sequences, with color-coded boxes representing the binding sites or transcription factors shown in the key. AP, activator protein; BRE, B-response element; CTCF, CCCTC binding factor; TCF, transcription factor; EBF, early B cell factor; IRF, interferon regulatory factor; NFκB, nuclear factor kappa light chain enhancer of activated B cells; PAX, paired box; PU, purine rich; SP, specificity protein; STAT, signal transducer and activator of proteins; TATA, thymidine adenine.

Figure 3

Interferon regulatory factor 5 promoter activity in immune cells. The luciferase plasmids were transfected by electroporation of three cell types: LCL, U937 cells, and Jurkat cells. A control GFP-encoding plasmid was also used in each sample to normalize transfection efficiency. ANOVA analysis revealed statistically significant variation between groups (p = 0.014); therefore t-tests were used to determine where the variation was found. The levels of transcription were significantly lower in Jurkat cells compared to LCL and U937 cells (p < 0.01). IRF5 is not highly expressed in T cells such as Jurkat cells, but is normally expressed in B cells and monocytes (39). The 1A promoters (1A_risk and 1A_protective) displayed higher activity than the 1B, 1C, or 1D promoters in LCL and Jurkat cells (p = 0.0009 and p = 0.016, respectively). In LCLs the 1A_risk promoter activity was higher than 1A_protective promoter activity (p = 0.019). The putative 1B promoter acted like the 1A promoter in that expression was significantly higher in LCLs than in Jurkat cells (p = 0.027). LCL, lymphoblastoid cell line; RLU, relative luminescence units; RFU, relative fluorescence units. In some samples, the first exon was not detectable, which is why there is some variation in sample number.

Figure 4

Imiquimod stimulates LCLs to express cytokines and interferon-response genes. LCLs were generated by EBV infection of cells from healthy volunteers. Cells were treated with imiquimod. (A) Expression of the interferon-response genes CCR7, NOXA, and Calreticulin were measured using SYBR Green real-time PCR. The figure shows the fold increase in gene expression after imiquimod stimulation for each gene. CCR7 and NOXA were significantly upregulated after imiquimod stimulation (p = 0.008 and p = 0.003, respectively). (B) Expression of cytokine RNA was measured using SYBR Green real-time PCR. IL-6 expression was upregulated by 71-fold after imiquimod stimulation (p = 0.028). IL-10 expression was also significantly upregulated (p = 0.038), although to a much lesser extent, at 1.5-fold. N = 12 for each experiment.

Figure 5

Imiquimod caused increased IRF5 transcription through exons 1A and 1D. All mRNA levels were measured in LCLs generated from healthy individuals. Levels were determined by TaqMan-based quantitative PCR using the 2^−ΔΔCT method. (A) The levels of IRF5 were 1.9-fold higher in treated cells (p = 0.0002). (B) The levels of exon 1A increased 2.2-fold (p = 0.030) and exon 1D increased by 2.8-fold (p = 0.033). All fold-increase values were normalized to the β-GUS housekeeping gene. The numbers in parentheses indicate the sample size. Analysis of variance was performed including each first exon and stimulation state as groups. This analysis revealed statistically significant variation (p < 0.0001). Statistical significance between individual groups was determined by paired t-test. IRF, interferon regulatory factor.

Figure 6

The 2004640 risk allele affects responsiveness to TLR7 stimulation. (A) LCLs with either risk or protective genotypes were treated with imiquimod to stimulate TLR7. Expression of interferon-response genes before and after stimulation was compared using SYBR Green real-time PCR. RNA input was normalized for each sample using the housekeeping gene GAPDH. Increase after imiquimod stimulation for risk and protective cells is shown. IRF5 expression is higher after imiquimod stimulation in the risk cells, by 1.67-fold (p = 0.021). Although Calreticulin expression decreases in both the risk and protective cells, it does so less in the risk cells (p-0.05). CCR7 increases less in the risk cells after imiquimod treatment (p = 0.05) N = 12 (B). EBV status does not dramatically effect IRF5 levels. IRF5 expression was compared between LCLs and Ramos cells, a Burkitt’s lymphoma-derived B cell line that is EBV negative. IRF5 levels were not dramatically different between cell linesN = 2.

Figure 7

Interferon regulatory factor 5 1B promoter activity is not regulated by direct p53 binding. (A) Different LCLs generated from healthy volunteers were electroporated with the 1B_wt or the 1B_p53* promoter luciferase plasmid. Cells were treated with 0.5 mM etoposide or left untreated. Despite the presence of a putative p53 binding site, DNA damaging treatment did not affect promoter activity; neither did mutation of the p53 site. (B) The putative p53 binding site in IRF5’s exon 1B promoter, with a WebLogo of the p53 consensus binding site (29) to indicate important bases and matches. The height of the base represents the frequency of that nucleotide. Site-directed mutagenesis was performed to mutate the binding site at the critical C and G bases as shown. UV, ultraviolet; wt, wild type; *, mutant.