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

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.

mTOR and other effector kinase signals that impact T cell function and activity

“…Interestingly, splice variants in RasGRP1 have been identified in human systemic lupus erythematosus patients. Additionally, variants in RasGRP1 have been associated with type 1 diabetes and Graves' disease, and loss of precise regulation of RasGRP1 expression levels appear to underlie the autoimmune disease in some of these patients …”

Lupus Susceptibility Region Containing CDKN1B rs34330 Mechanistically Influences Expression and Function of Multiple Target Genes, Also Linked to Proliferation and Apoptosis

“…A DNA pull‐down assay was performed as previously described (24). Briefly, nuclear extract from cultured Jurkat cell lines was incubated with biotin‐labeled DNA (risk and non‐risk alleles of rs34330) attached to Dynabeads M‐280.…”

“…Cells were harvested for RNA 72 hours post‐transfection. RNA was extracted, and qPCR was performed as previously described (24).…”

Alternative Splicing: A New Cause and Potential Therapeutic Target in Autoimmune Disease

“…SRSF1 knockdown in healthy human T cells via small interfering RNAs (siRNAs) increased the splice variant A:normal RasGRP1 isoform ratio and downregulated RasGRP1 (60). Moreover, hnRNP-K protein was correlated with RASGRP1 expression and extracellular signal-regulated kinase (ERK) phosphorylation (61).…”