Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression

Abstract

Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1.

Figure 1

ETS1-Imputed Genetic Variants Demonstrate Genome-wide Lupus Association in a Cohort of Asian Ancestry Each variant is represented as a data point in the context of its genomic location and is colored on the basis of whether it was directly genotyped (red) or only imputed (blue). Genomic position is given with GRCh37 coordinates. rs6590330 was directly genotyped. The SLE association of genotyped and imputed variants in cohorts of Asian and Asian-American (AS) ancestry (12,57 case and 1,258 control subjects), Hispanic-American (HA) ancestry (952 case and 335 control subjects), African-American (AA) ancestry (1,524 case and 1,809 control subjects), and European and European-American (EU) ancestry (3,926 case and 3,490 control subjects) were assessed in a logistic regression with adjustment for admixture estimates. Genome-wide association was defined as p < 5 × 10⁻⁸ and is indicated by a dashed red line in each figure panel.

Figure 2

A Single Genetic Effect Marked by Genotyped SNV rs6590330 Contributes to Lupus Risk in the AS Cohort Genomic position is given with GRCh37 coordinates. (A) The logistic regression association of genotyped variants in an AS cohort with an adjustment for admixture. (B) The logistic association of genotyped variants in an AS cohort with an adjustment for admixture and rs6590330. (C) The logistic association of genotyped and imputed variants in an AS cohort with an adjustment for admixture. (D) The logistic association of genotyped and imputed variants in an AS cohort with an adjustment for admixture and rs6590330.

Figure 3

Bayesian Association Plot Showing the Signal Strength in ETS1 as the Posterior Probability of Each SNV Genomic position is given with GRCh37 coordinates; AS data are shown. SNVs are colored according to their origin: genotyped variants are in red, and imputed variants are in blue. Variants in the 95% credible set are marked by diamonds. Variants with larger posterior probabilities (>0.01) represent those most likely to be causal.

Figure 4

The Lupus Risk Allele of rs6590330 Increases STAT1 Binding (A) STAT1 and pSTAT1 exhibit higher binding to oligonucleotides containing the rs6590330 risk allele than to oligonucleotides containing the non-risk allele. Biotin-labeled oligonucleotides were incubated with nuclear extract from Epstein-Barr-virus-transformed B cells. Proteins bound to the oligonucleotides were captured with the μMACs Factor Finder Kit. Proteins were then separated by SDS-PAGE and detected with anti-pSTAT1 (top) and anti-STAT1 (bottom). Abbreviations are as follows: M, marker; NR, oligonucleotide containing the non-risk allele of rs6590330; R, oligonucleotide containing the risk allele of rs6590330 (see Figure S5); mutant, oligonucleotide containing a disrupted putative STAT binding site downstream of rs6590330; and cell lysate, nuclear extract from B cells. The relative intensities of the bands are given above each band. Results are representative of four experiments; although all experiments demonstrated increased STAT1 binding to the probes with the risk allele, in two of four experiments, no STAT1 or pSTAT1 was detected in the immunoprecipitate from the non-risk oligonucleotide, whereas both were detected in the immunoprecipitate from the risk oligonucleotide. (B) rs6590330-heterozygous Epstein-Barr-virus-transformed B cells were used for ChIP-qPCR assessment of the differential binding of STAT1 to the risk and non-risk alleles. Crosslinked and sonicated chromatin was immunoprecipitated with an anti-STAT1 antibody. Site-specific primers and probes specific to the rs6590330 risk and non-risk alleles were used for determining STAT1 binding to immunoprecipitated DNA.