Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response

Abstract

Regulatory variants are often context specific, modulating gene expression in a subset of possible cellular states. Although these genetic effects can play important roles in disease, the molecular mechanisms underlying context specificity are poorly understood. Here, we identified shared quantitative trait loci (QTLs) for chromatin accessibility and gene expression in human macrophages exposed to IFNγ, Salmonella and IFNγ plus Salmonella. We observed that ~60% of stimulus-specific expression QTLs with a detectable effect on chromatin altered the chromatin accessibility in naive cells, thus suggesting that they perturb enhancer priming. Such variants probably influence binding of cell-type-specific transcription factors, such as PU.1, which can then indirectly alter the binding of stimulus-specific transcription factors, such as NF-κB or STAT2. Thus, although chromatin accessibility assays are powerful for fine-mapping causal regulatory variants, detecting their downstream effects on gene expression will be challenging, requiring profiling of large numbers of stimulated cellular states and time points.

Figure 1. Regulation of gene expression in macrophage immune response.

(a) Genetic variant has a direct effect on the binding of a stimulation-specific TF (IRF1) and target gene activation. (b) Genetic variant in a primed enhancer disrupts the binding of a cell type specific TF (e.g. PU.1) that indirectly influences stimulation-specific TF (IRF1) binding via modulation of chromatin accessibility. (c) Overview of the experimental design. (d) TLR4 recognises lipopolysaccharide (LPS) on Salmonella cell wall and activates NF-κB, AP-1 and IRF3 transcription factors (TFs). IRF3 stimulates IFNβ production that culminates with the activation of STAT1-STAT2-IRF9 complex. IFNγ binds to IFNγ receptor and activates STAT1 and IRF1 TFs.

Figure 2. Quantifying the extent of enhancer priming in macrophage immune response.

(a) Clustering of response eQTLs by effect size. Response eQTLs appear after IFNγ stimulation (clusters 5 and 6), Salmonella infection (clusters 2-4) or only when both of the stimuli are present (cluster 1). Relative effect was calculated by dividing the log₂ fold change values in each condition by the maximal fold change observed across conditions. (b) Quantile normalised log₂ fold changes of eQTL-caQTL pairs in naive and stimulated conditions. The pairs are grouped by the condition in which the eQTL had the largest effect (I, S or I+S). The heat maps are sorted by caQTL effect size in the naive condition (first column). The solid lines represent the 1.5-fold threshold above which the caQTLs are considered to be present in the naive condition. (c) Comparison of our estimated rate of enhancer priming (caQTL precedes response eQTL) to a negative control (eQTL precedes a response caQTL). (d) Association between rs4486968 variant, chromatin accessibility and and gene expression (n = 84 independent donors) in the GP1BA locus. (e) Association between rs7594476 variant, chromatin accessibility and gene expression (n = 84 independent donors) in the NXPH2 locus. FPM, fragments per million. The - log₁₀ p-values on panels d and e were calculated using RASQUAL. The caQTL analysis used n = 42 (N), n = 41 (I) and n = 31 (I+S) independent donors. Box plots show the median (center line) and the 25th and 75th percentiles (box edges), with whiskers extending to 1.5 times the interquartile range.

Figure 3. Identifying caQTLs that regulate chromatin accessibility at multiple independent regions.

(a) Fine mapping the putative causal variant at the NXPH2 locus. The master caQTL region (E1) overlaps a PU.1 ChIP-seq peak. Only one of the two variants (rs7594476) within the E1 region is predicted to disrupt the PU.1 motif (M6119_1.02). No associated variants overlap the IFNγ-specific dependent region E2. (b) Classifying caQTLs into master regions (i), dependent regions (ii) and ambiguous cases where the credible set overlaps either multiple regulated regions (iii) or does not overlap any regulated regions (iv-v). (c) Histogram of the number of associated dependent regions for each master region. (d) Multiple open chromatin regions regulated by a single caQTL at the NXPH2 locus. Master caQTL region (E1) and two IFNγ-specific dependent regions (E2 and E3) are highlighted by grey shadows. FPM, fragments per million. (e) Association between rs7594476 variant and expression of NXPH2 and SPOPL genes before and after IFNγ stimulation (n = 84 independent donors). (f) Association between rs7594476 variant, master caQTL region (E1) and two dependent regions (E2 and E3) before and after IFNγ stimulation (n = 42 and n = 41 independent donors, respectively). The -log₁₀ p-values on panels a and d were calculated using RASQUAL. Box plots show the median (center line) and the 25th and 75th percentiles (box edges), with whiskers extending to 1.5 times the interquartile range.

Figure 4. Identifying eQTLs and caQTLs that colocalise with complex disease risk loci.

(a) Total number of colocalised GWAS hits identified for each trait across the four conditions. (b) Cumulative number of colocalised GWAS hits identified by starting with overlaps in the naive condition and sequentially adding IFNγ, Salmonella and IFNγ + Salmonella conditions. (c) Colocalisation between an SLE GWAS hit (rs11997338), chromatin accessibility and CTSB gene expression (n = 84 independent donors) before and after IFNγ + Salmonella stimulation. The caQTL results are based on n = 42 (naive) and n = 31 (IFNγ + Salmonella) independent donors. FPM, fragments per million. Disease acronyms: SLE, systemic lupus erythematosus; IBD, inflammatory bowel disease; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; AD, Alzheimer’s disease; SCZ, schizophrenia; T2D, type 2 diabetes; NAR, narcolepsy; CEL, celiac disease. The -log₁₀ p-values on panels c were calculated using FastQTL. Box plots show the median (center line) and the 25th and 75th percentiles (box edges), with whiskers extending to 1.5 times the interquartile range.