title: An enhancer element harboring variants associated with systemic lupus erythematosus engages the TNFAIP3 promoter to influence A20 expression.
abstract: Functional characterization of causal variants present on risk haplotypes identified through genome-wide association studies (GWAS) is a primary objective of human genetics. In this report, we evaluate the function of a pair of tandem polymorphic dinucleotides, 42 kb downstream of the promoter of TNFAIP3, (rs148314165, rs200820567, collectively referred to as TT>A) recently nominated as causal variants responsible for genetic association of systemic lupus erythematosus (SLE) with tumor necrosis factor alpha inducible protein 3 (TNFAIP3). TNFAIP3 encodes the ubiquitin-editing enzyme, A20, a key negative regulator of NF-κB signaling. A20 expression is reduced in subjects carrying the TT>A risk alleles; however, the underlying functional mechanism by which this occurs is unclear. We used a combination of electrophoretic mobility shift assays (EMSA), mass spectrometry (MS), reporter assays, chromatin immunoprecipitation-PCR (ChIP-PCR) and chromosome conformation capture (3C) EBV transformed lymphoblastoid cell lines (LCL) from individuals carrying risk and non-risk TNFAIP3 haplotypes to characterize the effect of TT>A on A20 expression. Our results demonstrate that the TT>A variants reside in an enhancer element that binds NF-κB and SATB1 enabling physical interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB to the TT>A risk alleles or knockdown of SATB1 expression by shRNA, inhibits the looping interaction resulting in reduced A20 expression. Together, these data reveal a novel mechanism of TNFAIP3 transcriptional regulation and establish the functional basis by which the TT>A risk variants attenuate A20 expression through inefficient delivery of NF-κB to the TNFAIP3 promoter. These results provide critical functional evidence supporting a direct causal role for TT>A in the genetic predisposition to SLE.
authors.affiliation: Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America.
authors.forename: Shaofeng
authors.initials: S
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Wen
authors.forename: Graham B
authors.initials: GB
authors.lastname: Wiley
authors.forename: Michael T
authors.initials: MT
authors.lastname: Kinter
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Gaffney
mesh_terms: D000483:Alleles; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006239:Haplotypes; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000209:etiology / Q000235:genetics* / Q000473:pathology; D036961:Matrix Attachment Region Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D036961:Matrix Attachment Region Binding Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; C076189:SATB1 protein, human; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
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