Background/Purpose:

Genome Wide Association studies (GWAS) have identified several variants in the tumor necrosis factor alpha-induced protein 3 (TNFAIP3) gene associated with autoimmune disease. TNFAIP3 encodes A20, an ubiquitin-editing enzyme that inhibits Nuclear Factor-kappa B (NF-κB) signaling to modulate innate and adaptive immune responses. Loss of TNFAIP3 expression is implicated in the pathogenesis of autoimmune disease; however, mechanisms responsible for impaired expression remain unclear. Encyclopedia of DNA Elements (ENCODE) and Chromatin Interaction analysis by paired-end Tag Sequencing (ChIA-PET) suggests TNFAIP3 expression is regulated through a long-range interaction with a putative upstream enhancer. In this study, we investigated the functional importance of a predicted SLE risk variant reference single nucleotide polymorphism (rs)10499197 situated near this enhancer.

Methods:

Electrophoretic Mobility Shift Assays (EMSA) and affinity purification of nuclear factors were performed to screen for specific proteins with altered affinity for the rs10499197 risk allele. Results were validated using Epstein-Barr Virus (EBV) transformed B cell lines carrying non-risk or risk genotypes by Chromatin Immuno-Precipitation (ChIP) followed by quantitative PCR. To investigate the regulatory function of the putative enhancer, we cloned the non-risk and risk alleles into a minimal promoter vector containing the luciferase gene, and performed luciferase assays on immune cells (Jurkat and Tamm-Horsfall Protein 1(THP-1).

Results:

The rs10499197 risk allele bound the nuclear-protein complex with reduced affinity in EBV-B cells and Jurkat T cells, and increased affinity in THP-1 monocytes, suggesting a cell-specific affect. Affinity purification and ChIP studies revealed reduced binding of the risk allele to NF-κB subunits, NF-kB p50 Subunit (p50) and NF-kB p65 Subunit (p65), and enhanced binding for the risk allele to transcription factors, E2 Transcription Factor1 (E2F1) and Cyclic adenosine monophosphate Responsive Element Binding protein1 (CREB1). E2F1 and CREB1 negatively regulate NF-κB signaling by disrupting NF-kB p50-p65 heterodimer formation, thereby affecting NF-κB-mediated transcription of TNFAIP3. Consistently, luciferase assays demonstrated that the regulatory element functions as an enhancer of TNFAIP3 expression and that the risk allele impaired enhancer activity.

Conclusion:

Results suggest that the SLE risk variant rs10499197 is likely a causal variant that disrupts the function of a putative enhancer upstream of TNFAIP3. Disrupting the enhancer may impair TNFAIP3 expression, enhance NF-κB signaling, and heighten immune responses in a cell type specific manner. Following our work on the TT>A enhancer, this is the second likely causal variant to impact TNFAIP3 expression. Future Chromatin Conformation Capture assay (3C) will determine how the upstream DNA element physically interacts with the TNFAIP3 promoter, and help establish a comprehensive mechanistic understanding for how TNFAIP3 transcriptional regulation is modulated by SLE risk alleles.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-sle-risk-variant-reference-single-nucleotide-polymorphism-rs10499197-upstream-of-tumor-necrosis-factor-alpha-induced-protein-3-tnfaip3-modulates-enhancer-function-and-tnfaip3-gene/