Systemic lupus erythematosus variants modulate the function of an enhancer upstream of TNFAIP3

Abstract

TNFAIP3/A20 is a prominent autoimmune disease risk locus that is correlated with hypomorphic TNFAIP3 expression and exhibits complex chromatin architecture with over 30 predicted enhancers. This study aimed to functionally characterize an enhancer ∼55 kb upstream of the TNFAIP3 promoter marked by the systemic lupus erythematosus (SLE) risk haplotype index SNP, rs10499197. Allele effects of rs10499197, rs58905141, and rs9494868 were tested by EMSA and/or luciferase reporter assays in immune cell types. Co-immunoprecipitation, ChIP-qPCR, and 3C-qPCR were performed on patient-derived EBV B cells homozygous for the non-risk or SLE risk TNFAIP3 haplotype to assess haplotype-specific effects on transcription factor binding and chromatin regulation at the TNFAIP3 locus. This study found that the TNFAIP3 locus has a complex chromatin regulatory network that spans ∼1M bp from the promoter region of IL20RA to the 3′ untranslated region of TNFAIP3. Functional dissection of the enhancer demonstrated co-dependency of the RelA/p65 and CEBPB binding motifs that, together, increase IL20RA and IFNGR1 expression and decreased TNFAIP3 expression in the context of the TNFAIP3 SLE risk haplotype through dynamic long-range interactions up- and downstream. Examination of SNPs in linkage disequilibrium (D’ = 1.0) with rs10499197 identified rs9494868 as a functional SNP with risk allele-specific increase in nuclear factor binding and enhancer activation in vitro. In summary, this study demonstrates that SNPs carried on the ∼109 kb SLE risk haplotype facilitate hypermorphic IL20RA and IFNGR1 expression, while suppressing TNFAIP3 expression, adding to the mechanistic potency of this critically important locus in autoimmune disease pathology.

3D genome organization and epigenetic regulation in autoimmune diseases

“…The gene TNFAIP3 is a likely target of the discovered enhancer, as it is a major negative regulator of pro-inflammatory nuclear factor kappa B (NF-κB) signaling, which is associated with many autoimmune diseases ( 137 ). Moreover, the risk allele (A/A) in the enhancer dramatically disrupts NF-κB binding and inhibits long-range DNA looping of the enhancer to the TNFAIP3 promoter, effectively decreasing TNFAIP3 expression and increasing IL20RA and IFNGR1 expression through TALEN (transcription activation-like effector nuclease)-mediated genome editing, HiChIP, 3C-qPCR, and dual-luciferase reporter assays ( 85 , 138 ). TNFAIP3 loss-of-function mutations result in elevated inflammatory responses, autoantibody production, and inflammatory arthritis in humanized mouse models ( 86 , 139 ).…”

A genome-wide CRISPR screen supported by human genetics identifies theTNRC18gene locus as a novel regulator of inflammatory signaling

“…For example, TNFAIP3 encodes the AP20 enzyme, a known regulator of NFkB-mediated immune activation 36 and pro-IL-1β processing 37,38 . The association signals at the TNFAIP3 gene locus have been resolved to TNFAIP3 using experimental [39][40][41] or in silico 42 methods for some of the immune-mediated diseases.…”

Current research into A20 mediation of allergic respiratory diseases and its potential usefulness as a therapeutic target

“…It acts as an endogenous negative regulator of NF-κB signaling and has anti-inflammatory and immunomodulatory effects in various inflammatory and autoimmune diseases ( 12 ). Single-nucleotide polymorphisms (SNPs) on the TNFAIP3 locus are correlated to several inflammatory disorders, including rheumatoid arthritis ( 12 , 13 ), systemic lupus erythematosus ( 14 ), and inflammatory bowel disease ( 15 ). A20 is involved in allergic disease pathogenesis ( 16 ).…”