Proteome-Wide Analysis of Disease-Associated SNPs That Show Allele-Specific Transcription Factor Binding

<div><p>A causative role for single nucleotide polymorphisms (SNPs) in many genetic disorders has become evident through numerous genome-wide association studies. However, identification of these common causal variants and the molecular mechanisms underlying these associations remains a major challenge. Differential transcription factor binding at a SNP resulting in altered gene expression is one possible mechanism. Here we apply PWAS (“proteome-wide analysis of SNPs”), a methodology based on quantitative mass spectrometry that enables rapid screening of SNPs for differential transcription factor binding, to 12 SNPs that are highly associated with type 1 diabetes at the <em>IL2RA</em> locus, encoding the interleukin-2 receptor CD25. We report differential, allele-specific binding of the transcription factors RUNX1, LEF1, CREB, and TFAP4 to <em>IL2RA</em> SNPs rs12722508*A, rs12722522*C, rs41295061*A, and rs2104286*A and demonstrate the functional influence of RUNX1 at rs12722508 by reporter gene assay. Thus, PWAS may be able to contribute to our understanding of the molecular consequences of human genetic variability underpinning susceptibility to multi-factorial disease.</p> </div

Schematic representation of a two-dimensional interaction plot.

<p>While specific outliers are found in the upper left (variant A) or the lower right (variant G) quadrant, most proteins cluster around the origin as they are binding to both variants equally. Contaminants have a SILAC ratios lower than 1 even when labels are switched and thus are grouped in the lower left quadrant.</p

Overview of all SNPs analyzed in this study with T1D disease association.

<p>SNPs which mark their respective haplotype are marked in bold.</p

SNP pull-down for TFAP2 and SP1.

<p>A: Coomassie stained and anti-TFAP2 immunostained blot. TFAP2 enrichment with the SELEX-derived binding site can be visualized by Western blot, but not within the context of other protein bands in Coomassie staining. B: SNP pull-down for TFAP2. The transcription factor binds to its SELEX sequence (C variant), but a single nucleotide exchange abrogates binding (A variant). Performing proteome-wide detection for differentially binding transcription factors, only TFAP2 is enriched at the wild-type sequence observed in the individual mass spectrum of the TFAP2 peptide LSLLSSTSK²⁺ and by comparison of all SILAC ratios of proteins detected in the experiment. C: SNP pull-down for rs509813. Applying PWAS, we identify SP1 together with SP3, which both bind to the same DNA motif. ZNF148, was also detected as a significant interaction partner in our proteome-wide screen, although not bioinformatically predicted.</p

Interaction analysis for fine-mapped T1D SNPs.

<p>Two-dimensional interaction plots reveal the specific binding of transcription factors. A: CREB1 and TFAP4 are enriched on rs12722522*C while this allele resembles a perfect match to the CREB consensus binding motif. B: rs12722508 is bound by several transcription factors (upper panel) among them the RUNX1-CBFB heterodimer, although the sequence around rs12722508*A does not match perfectly the transcription factor consensus motif (lower panel); C: Quantitative SNP pull-down screen identifies LEF1 to bind around 8 times stronger to rs41295061*A.</p

Schematic diagram of SNP pull-down.

<p>Synthetic oligonucleotides containing the SNP are phosphorylated, polymerized and subsequently strand-specifically desthiobiotin-labeled. The immobilized DNA fragments are incubated with either light or heavy extract. After removal of unbound proteins, bead fractions are combined and DNA-protein complexes are eluted with biotin. The eluate is precipitated, digested and analyzed by single-run, high resolution, quantitative mass spectrometry. Specific interaction partners result in a ratio different from 1∶1, demonstrating specific enrichment at one variant of the single nucleotide polymorphism.</p

Functional analysis for SNP rs12722508.

<p>A: Immunostaining with an antibody against endogenous RUNX1 validates differential binding between the A- and G-allele of rs12722508, input refers to nuclear extract incubated with either rs12722508 allele. B: mRNA levels are reduced upon esiRNA knock-down with mean and s.d. of triplicates; C: changes in firefly luciferase activity in mock-transfection and upon knock-down of TFAP4, RUNX1, CREB and TFAP4. Knock-down of RUNX1 results in an activation with an allele-specific difference (<i>P</i> = 0.016) demonstrating the functional consequence of differential RUNX1 binding between the two alleles of rs12722508.</p

data_sheet_1_Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages.PDF

<p>GPR84 is a member of the metabolic G protein-coupled receptor family, and its expression has been described predominantly in immune cells. GPR84 activation is involved in the inflammatory response, but the mechanisms by which it modulates inflammation have been incompletely described. In this study, we investigated GPR84 expression, activation, and function in macrophages to establish the role of the receptor during the inflammatory response. We observed that GPR84 expression in murine tissues is increased by endotoxemia, hyperglycemia, and hypercholesterolemia. Ex vivo studies revealed that GPR84 mRNA expression is increased by LPS and other pro-inflammatory molecules in different murine and human macrophage populations. Likewise, high glucose concentrations and the presence of oxidized LDL increased GPR84 expression in macrophages. Activation of the GPR84 receptor with a selective agonist, 6-(octylamino) pyrimidine-2,4(1H,3H)-dione (6-n-octylaminouracil, 6-OAU), enhanced the expression of phosphorylated Akt, p-ERK, and p65 nuclear translocation under inflammatory conditions and elevated the expression levels of the inflammatory mediators TNFα, IL-6, IL-12B, CCL2, CCL5, and CXCL1. In addition, GPR84 activation triggered increased bacterial adhesion and phagocytosis in macrophages. The enhanced inflammatory response mediated by 6-OAU was not observed in GPR84^−/− cells nor in macrophages treated with a selective GPR84 antagonist. Collectively, our results reveal that GPR84 functions as an enhancer of inflammatory signaling in macrophages once inflammation is established. Therefore, molecules that antagonize the GPR84 receptor may be potential therapeutic tools in inflammatory and metabolic diseases.</p