Background/Purpose: Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with complex genetic underpinnings. Variants from RASGRP3 (RAS Guanyl Releasing Protein 3) is one of the most consistently replicated SLE susceptibility genes. We recently reported that two intronic variants (rs13385731 and rs12612030) explained RASGRP3-SLE association in Asians [Sun et al. 2016, Nat Genet]. However, the “causal” functional variants and the genetic mechanism(s) by which associated variants contribute to disease are largely unknown. We hypothesized that these intronic variants affect epigenetic regulation and modulate RASGRP3 expression.

Methods: First, we used in-silico bioinformatics and epigenetic analyses to define the potential regulatory effects of the candidate variants on gene expression using data on several histone marks, DNAse-1 hypersensitivity, and eQTLs across multiple tissues from ENCODE, ROADMAP and GTEx data bases. Luciferase reporter assays in HEK293 cells were used to measure the effects of risk and non-risk variants/haplotypes on gene expression. Next, we used a combination of DNA pulldown, Electrophoretic Mobility Shift Assay (EMSA), Super-shift, Western blot and Mass Spectrometry to identify DNA-bound proteins followed by quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) to assess the allele-specific binding of interacting proteins. To assess the effect of risk alleles/haplotypes on cis-regulatory elements we also performed ChIP-qPCR with H3K27Ac and P300. Finally, using EBV-transformed cell lines, RASGRP3 mRNA and protein expressions were compared between risk and non-risk alleles/haplotypes.

Results: Bioinformatics predicted that these variants are located in active chromatin and have the potential to be dual enhancers/promoters. We also predicted allele-specific binding to PARP1 and IRF1 at rs13385731. We observed significant (p<0.005) difference in RASGRP3 transcript and protein levels with increased expression in rs13385731 risk genotype (TT). Luciferase assays demonstrated significant (p<0.005) allele-specific enhancer and promoter activities. DNA pulldown and EMSA suggested allele-specific bound protein ~100 kD, which was identified as PARP1 protein by Mass Spectrometry and later confirmed by super shift and Western blot. We also verified differential allele-specific binding of PARP1 and IRF1 against rs13385731 using ChIP-qPCR. Interestingly, while PARP1 binding affinity is higher with risk (TT) genotype, IRF1 binding shows strong binding affinity with non-risk (CC) genotype of rs13385731.

Conclusion: Our results showed that rs13385731 is an eQTL, and the risk (TT) genotype is associated with increased RASGRP3 expression. Furthermore, this variant showed significant allele-specific binding to H3K27Ac, P300, PARP1 and IRF1 proteins, which might alter expression of RASGRP3, contributing to SLE risk. The activity of this variant provides insight into the molecular mechanisms underlying its association with SLE.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/intronic-variants-of-the-b-cell-proliferator-rasgrp3-affect-its-expression-and-might-contribute-to-lupus-risk/