Background: GWAS have identified more than 80 susceptibility loci associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. Fine mapping of the TNFAIP3 interacting protein 1 (TNIP1) locus identified two independent TNIP1 SLE risk haplotypes that reduced the expression of TNIP1 mRNA and ABIN1 protein. In this work, we characterized SLE-associated candidate causal SNPs that influence hypomorphic expression of TNIP1.
Methods: We selected 11 SLE-associated TNIP1 SNPs with low binding scores (RegulomeDB) for EMSAs to evaluate whether SLE risk alleles affect binding of nuclear protein complexes extracted from different immune cells treated with and without PMA/Ionomycin (P/I). Affinity DNA pull-down assays and Western blotting (WB) were performed to identify proteins bound to the rs10036748 probe. Enhancer activity of rs10036748 was measured by luciferase assay.
Results: Three of the elevenTNIP1 risk variants exhibited reduced binding of nuclear proteins from Jurkat T cells, EBV transformed B cells and THP-1 monocytoid cells. Five risk variants demonstrated P/I stimulation dependency. Furthermore, three risk variants showed increased binding under resting conditions; one showed increased binding after P/I stimulus. Overall, the SNPs carried on both TNIP1 SLE risk haplotypes demonstrated complex binding activity; Jurkat T cells exhibited the most activity with 8 of 11 SNPs showing differential binding. The rs10036748 variant demonstrated reduced binding of nuclear proteins to the risk allele in all cell types. Affinity pull-down followed by WB confirmed ENCODE-defined transcription factor binding predictions that the rs10036748 non-risk allele bound early growth response protein 1 (EGR-1), cyclic AMP-responsive element binding protein 1 (CREB-1), and class E basic helix-loop-helix protein 40 (bHLHe40) with relative higher affinity than the risk allele. Interestingly, the risk allele showed increased enhancer activity in HEK293T, Jurkat and EBV transformed B cells.
Conclusion: Functional analyses of SNPs in TNIP1 SLE risk haplotypes suggest a complex regulation at TNIP1 locus. Further, these SNPs exhibit cell type specific, stimulation dependent and allele specific binding of transcriptional protein complexes. Regulatory insights gained will better direct future characterization of individual SLE-associated TNIP1 variants in-vivo to decipher molecular mechanisms and cell states that contribute to SLE pathogenesis.