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

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. © 2013 wang et al.