Session Type: ACR Concurrent Abstract Session
Session Time: 11:00AM-12:30PM
Background/Purpose: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by autoantibody production and dysregulated interferon responses. Genome-wide association studies (GWAS) have identified more than 80 susceptibility or risk genes/loci associated with SLE and other autoimmune diseases, including TNFAIP3 interacting protein 1 (TNIP1). TNIP1 encodes the adaptor protein ABIN1, which functions in the immune system by restricting NF-κB signaling. Previous fine mapping of the TNIP1 locus demonstrated significant associations with SLE and single nucleotide polymorphisms (SNPs) that are carried on two independent risk haplotypes within TNIP1. Both risk haplotypes demonstrated reduced expression of TNIP1 mRNA and ABIN1 protein. In this work we characterize associated SNPs in TNIP1 and identify candidate causal SNPs that influence hypomorphic expression of TNIP1.
Methods: Eleven TNIP1 SNPs from both SLE risk haplotypes which have lower binding scores (RegulomeDB) were selected for electrophoretic mobility shift assays (EMSA) to evaluate whether SLE risk alleles affect binding of nuclear protein complexes extracted from different immune cells that were treated with and without PMA/Ionomycin (P/I) in 3 independent EMSA tests. Affinity DNA pull-down assay and Western blotting (WB) was performed to identify proteins bound to rs10036748 probe.
Results: EMSAs using probes containing non-risk and risk alleles of 11 SLE associated SNPs in the TNIP1 locus identified 3 variants with reduced binding of nuclear proteins to the SLE risk allele from the Jurkat T cell line, EBV transformed B cells and the monocytoid cell line, THP-1. Five of the 11 variants demonstrated P/I stimulation dependency of which 2 variants showed decreased binding to the risk allele and 3 variants showed increased binding to the risk allele. Interestingly, 3 TNIP1 SNPs showed increased binding of nuclear proteins to the risk-allele under resting conditions; one of these was P/I stimulus dependent. Overall, the SNPs carried on both TNIP1 SLE risk haplotypes demonstrated complex binding activity with the Jurkat T cell line having the most activity (8 of 11 SNPs showing differential binding). To begin verifying the identity of the nuclear proteins present in the EMSA, we used affinity pull-down followed by WB for the rs10036748 variant which had reduced binding of nuclear proteins to risk-allele in all 3 cell types. Based on ENCODE defined transcription factor binding, we confirm that early growth response 1 and NF-κB subunit, RelA are associated with this variant DNA and reduced with the risk allele of rs10036748.
Conclusion: Functional analyses of SNPs in TNIP1 SLE risk haplotypes suggested a complex regulation at TNIP1 locus. The association of transcriptional protein complexes at these SNPs is highly regulated showing cell type specificity, stimulation dependency and allele specific binding. The regulatory insights gained through in-vitro assays will better direct us to further separate and characterize individual SLE associated TNIP1 variants in-vivo to decipher molecular mechanisms and cell states by which TNIP1 risk haplotypes contribute to SLE pathogenesis.
To cite this abstract in AMA style:Pasula S, Wiley M, Wu YY, Nair A, Gopalakrishnan J, Gaffney PM. Identification and Functional Characterization of TNIP1 Causal Variants Associated with Systemic Lupus Erythematosus [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/identification-and-functional-characterization-of-tnip1-causal-variants-associated-with-systemic-lupus-erythematosus/. Accessed December 5, 2020.
« Back to 2016 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-and-functional-characterization-of-tnip1-causal-variants-associated-with-systemic-lupus-erythematosus/