@article {64973, title = {Mechanistic characterization of RASGRP1 variants identifies an hnRNP K-regulated transcriptional enhancer contributing to SLE susceptibility.}, journal = {Frontiers in Immunology}, year = {2019}, month = {2019 May 20}, abstract = {

Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component. We recently identified a novel SLE susceptibility locus near\ RASGRP1, which governs the ERK/MAPK kinase cascade and B-/T-cell differentiation and development. However, precise causal\ RASGRP1functional variant(s) and their mechanisms of action in SLE pathogenesis remain undefined. Our goal was to fine-map this locus, prioritize genetic variants likely to be functional, experimentally validate their biochemical mechanisms, and determine the contribution of these SNPs to SLE risk. We performed a meta-analysis across six Asian and European cohorts (9,529 cases; 22,462 controls), followed by\ in silico\ bioinformatic and epigenetic analyses to prioritize potentially functional SNPs. We experimentally validated the functional significance and mechanism of action of three SNPs in cultured T-cells. Meta-analysis identified 18 genome-wide significant (p\ \< 5 {\texttimes} 10-8) SNPs, mostly concentrated in two haplotype blocks, one intronic and the other intergenic. Epigenetic fine-mapping, allelic, eQTL, and imbalance analyses predicted three transcriptional regulatory regions with four SNPs (rs7170151, rs11631591-rs7173565, and rs9920715) prioritized for functional validation. Luciferase reporter assays indicated significant allele-specific enhancer activity for intronic rs7170151 and rs11631591-rs7173565 in T-lymphoid (Jurkat) cells, but not in HEK293 cells. Following up with EMSA, mass spectrometry, and ChIP-qPCR, we detected allele-dependent interactions between heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and rs11631591. Furthermore, inhibition of hnRNP-K in Jurkat and primary T-cells downregulated\ RASGRP1\ and ERK/MAPK signaling. Comprehensive association, bioinformatics, and epigenetic analyses yielded putative functional variants of\ RASGRP1, which were experimentally validated. Notably, intronic variant (rs11631591) is located in a cell type-specific enhancer sequence, where its risk allele binds to the hnRNP-K protein and modulates\ RASGRP1\ expression in Jurkat and primary T-cells. As risk allele dosage of rs11631591 correlates with increased\ RASGRP1\ expression and ERK activity, we suggest that this SNP may underlie SLE risk at this locus.

}, doi = {10.3389/fimmu.2019.01066}, author = {Molineros, Julio E. and Singh, Bhupinder and Terao, Chikashi and Okada, Yukinori and Kaplan, Jakub and McDaniel, Barbara and Akizuki, Shuji and Sun, Celi and Webb, Carol F. and Looger, Loren L and Nath, Swapan K.} }