Promoter Polymorphisms in Interferon Regulatory Factor 5

• Main Author: Clark, Daniel N.
• Format: Others
• Published: BYU ScholarsArchive 2013
• Subjects: autoimmunity; cytokines; apoptosis; Microbiology
• Online Access: https://scholarsarchive.byu.edu/etd/4058; https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=5057&context=etd

The promoter region of interferon regulatory factor 5 (IRF5) contains the rs2004640 T or G single nucleotide polymorphism (SNP) and a CGGGG indel. Both of these polymorphisms have been implicated as genetic risk factors for several autoimmune diseases, including systemic lupus erythematosus, whose pathology involves altered apoptosis and cytokine signaling. The polymorphisms' overall effect is to increase IRF5 levels. IRF5 is a transcription factor of several cytokines, including interferon, and is pro-apoptotic. Thus an alteration of cytokine levels and apoptosis signaling due to high IRF5 levels is the proposed source of autoimmune risk. Each of IRF5's four first exons (1A, 1B, 1C, 1D) has its own promoter and responds to specific stimuli. rs2004640 is a T or G polymorphism; T is the risk allele. The SNP creates a sequence-specific recognition site for the spliceosome, making exon 1B spliceable. Analysis of the 1B promoter showed putative p53 binding site. IRF5 and p53 are pro-apoptotic transcription factors, and the p53 site may provide a positive feedback loop. Apoptosis levels were altered in cells with the rs2004640 risk T/T allele when treated with DNA damaging agents (extrinsic apoptosis), but not when activating death receptors (intrinsic apoptosis). The 1B promoter was the only one to activate expression after inducing DNA damage in a luciferase reporter assay, and this activation was abolished after mutating the p53 site. The exon 1A promoter contains either three or four copies (4X) of CGGGG; the 4X variant is the risk allele. The 1A promoter is constitutively active and is responsive to the Toll-like receptor 7 agonist imiquimod. RNA folding analysis revealed a hairpin encompassing exon 1B. Mutational analysis showed that the hairpin shape decreased translation five-fold in a luciferase reporter assay. Cells with the CGGGG or rs2004640 risk allele exhibited higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR in cell lines with either risk polymorphism demonstrated decreased usage of exons 1C or 1D, although no other correlated splicing events were observed. Also, several mRNA splice variants of IRF5 were sequenced. The risk polymorphisms altered cytokine signaling as well. Expression of interferon, Toll-like receptor, and B cell receptor pathways were affected by a risk haplotype which includes the rs2004640 SNP. The CGGGG polymorphism decreased the levels of CC-chemokine receptor 7. Specific transcription factor binding sites define promoter activity and thus first exon usage and transcription levels. Translation levels are affected by mRNA folding. Overall, the rs2004640 SNP and the CGGGG indel cause high levels of IRF5. High IRF5 expression causes altered cytokine and apoptosis signaling, and may bias the immune system toward autoimmunity.