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.通过全基因组关联研究（Genome-Wide Association Studies, GWAS）鉴定的风险单倍型上的致病变异的功能表征，是人类遗传学的核心研究目标之一。本研究评估了TNFAIP3启动子下游42 kb处的一对串联多态性二核苷酸（rs148314165、rs200820567，统称为TT>A）的功能，该变异近期被认定为介导系统性红斑狼疮（systemic lupus erythematosus, SLE）与肿瘤坏死因子α诱导蛋白3（tumor necrosis factor alpha inducible protein 3, TNFAIP3）遗传关联的致病变异。TNFAIP3编码泛素编辑酶A20，后者是核因子κB（NF-κB）信号通路的关键负调控因子。携带TT>A风险等位基因的受试者中，A20的表达水平显著降低，但其背后的分子机制尚未明确。本研究结合电泳迁移率变动分析（electrophoretic mobility shift assays, EMSA）、质谱（mass spectrometry, MS）、报告基因实验、染色质免疫沉淀-聚合酶链反应（chromatin immunoprecipitation-PCR, ChIP-PCR），以及来自携带风险与非风险TNFAIP3单倍型个体的EB病毒转化淋巴母细胞系（EBV transformed lymphoblastoid cell lines, LCL）的染色质构象捕获（chromosome conformation capture, 3C）技术，表征了TT>A变异对A20表达的调控作用。研究结果显示，TT>A变异位于一个结合NF-κB与SATB1的增强子元件中，该增强子可通过远距离DNA环化与TNFAIP3启动子发生物理互作。NF-κB与TT>A风险等位基因的结合能力受损，或通过短发夹RNA（short hairpin RNA, shRNA）敲低SATB1的表达，均会抑制该环化互作，进而导致A20表达下调。综上，本研究揭示了TNFAIP3转录调控的全新机制，并阐明了TT>A风险变异通过降低NF-κB向TNFAIP3启动子的有效递送，从而削弱A20表达的功能基础。上述结果为TT>A变异在系统性红斑狼疮遗传易感性中的直接致病作用提供了关键的功能学证据。