An enhancer element harboring variants associated with systemic lupus erythematosus engages the TNFAIP3 promoter to influence A20 expression.

 

ÀúÀÚ: Wang S, Wen F, Wiley GB, Kinter MT, Gaffney PM.

Àú³Î: PLoS Genet. 2013 Sep;9(9):e1003750

»ç¿ëÇÑ Á¦Ç°: Dynabeads M-280 Streptavidin (Cat# 11205D, 11206D, 60210)

 

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-¥êB 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-¥êB and SATB1 enabling physical interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-¥êB 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-¥êB to the TNFAIP3 promoter. These results provide critical functional evidence supporting a direct causal role for TT>A in the genetic predisposition to SLE.

 

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Affinity purification of nuclear factors

We screened for other proteins that bind to the EMSA probes by biotinylating the oligonucleotides used for EMSA and a scrambled oligonucleotide that served as a negative control (Table S1). Streptavidin magnetic beads (200 ug; Dynalbeads M-280 Streptavidin; Invitrogen) were subjected to two rounds of blocking with 1% BSA in PBS for 15 min and washing with PBS containing 1M NaCl and TE buffer. Biotinlyated oligonucleotides were linked to half the amount of streptavidin beads by incubating for 30 min at room temperature in TE buffer followed by washing with TE buffer. To pre-clear the nuclear extracts of material that could bind non-specifically to the biotinylated oligonucleotides, we incubated the other half of the BSA-blocked beads with 100 ug of nuclear extract in binding buffer (250 mM NaCl, 50 mM Tris Cl, 50% glycerol, 2.5 mM DTT, 2.5 mM EDTA, pH 7.6) containing 15 ng/ul poly dI:dC (Sigma-Aldrich), 0.5 ug/ml BSA, and 0.1% NP40 for 30 min on ice. We then incubated the pre-cleared nuclear extracts with the oligonucleotide-linked Streptavidin beads for 30 min in 37uC water bath with gentle shaking every 5 min, and subsequently washed the products with binding buffer containing 0.1% NP40 three times. The proteins were eluted in 50 ul of 0.2% SDS sample buffer by boiling for 5 min and were then resolved on a Nu-PAGE 4%–12% Bis-Tris gel followed by silver nitrate staining.

 

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