An Enhancer Element Harboring Variants Associated with Systemic Lupus Erythematosus Engages the TNFAIP3 Promoter to Influence A20 Expression

• Published in: PLoS genetics Vol. 9; no. 9; p. e1003750
• Main Authors: Wang, Shaofeng, Wen, Feng, Wiley, Graham B., Kinter, Michael T., Gaffney, Patrick M.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.09.2013; Public Library of Science (PLoS)
• Subjects: Alleles; Autoimmune diseases; Binding sites; Disease; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Editing; Enhancer Elements, Genetic; Enzymes; Experiments; Gene expression; Gene Expression Regulation; Genetic aspects; Genetic Predisposition to Disease; Genetic variation; Genomes; Haplotypes; HEK293 Cells; Homeostasis; Humans; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Kinases; Lupus; Lupus Erythematosus, Systemic - etiology; Lupus Erythematosus, Systemic - genetics; Lupus Erythematosus, Systemic - pathology; Mass spectrometry; Matrix Attachment Region Binding Proteins - antagonists & inhibitors; Matrix Attachment Region Binding Proteins - genetics; Matrix Attachment Region Binding Proteins - metabolism; NF-kappa B - genetics; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Physiological aspects; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Promoters (Genetics); Signal Transduction; Systemic lupus erythematosus; Tumor Necrosis Factor alpha-Induced Protein 3; United States
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1003750

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.