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Front Immunol. 2019 May 20;10:1066. doi: 10.3389/fimmu.2019.01066. eCollection 2019.

Mechanistic Characterization of RASGRP1 Variants Identifies an hnRNP-K-Regulated Transcriptional Enhancer Contributing to SLE Susceptibility.

Author information

1
Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, United States.
2
Laboratory for Statistical Analysis, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
3
Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
4
Department of Statistical Genetics, Osaka University Graduate School of Medicine, Osaka, Japan.
5
Departments of Medicine, Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma, OK, United States.
6
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, United States.

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 RASGRP1 functional 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 × 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.

KEYWORDS:

ChIP-qPCR; EMSA (electrophoretic mobility shift assay); ERK (extracellular-signal-regulated kinase); RASGRP1; genetic variant; homology; luciferase

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