Long-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene

Abstract

The chromosome 16p13 region has been associated with several autoimmune diseases, including type 1 diabetes (T1D) and multiple sclerosis (MS). CLEC16A has been reported as the most likely candidate gene in the region, since it contains the most disease-associated single-nucleotide polymorphisms (SNPs), as well as an imunoreceptor tyrosine-based activation motif. However, here we report that intron 19 of CLEC16A, containing the most autoimmune disease-associated SNPs, appears to behave as a regulatory sequence, affecting the expression of a neighbouring gene, DEXI. The CLEC16A alleles that are protective from T1D and MS are associated with increased expression of DEXI, and no other genes in the region, in two independent monocyte gene expression data sets. Critically, using chromosome conformation capture (3C), we identified physical proximity between the DEXI promoter region and intron 19 of CLEC16A, separated by a loop of >150 kb. In reciprocal experiments, a 20 kb fragment of intron 19 of CLEC16A, containing SNPs associated with T1D and MS, as well as with DEXI expression, interacted with the promotor region of DEXI but not with candidate DNA fragments containing other potential causal genes in the region, including CLEC16A. Intron 19 of CLEC16A is highly enriched for transcription-factor-binding events and markers associated with enhancer activity. Taken together, these data indicate that although the causal variants in the 16p13 region lie within CLEC16A, DEXI is an unappreciated autoimmune disease candidate gene, and illustrate the power of the 3C approach in progressing from genome-wide association studies results to candidate causal genes.

Figure 1.

Expression of the DEXI gene, contained within the 16p13 region of T1D association (A) by microarray using Illumina probe set ILMN_1738866, is correlated with the genotype at SNPs within the CLEC16A gene in two independent data sets. The first data set (i) was generated from purified human monocytes (n= 1370 individuals) collected during GHS and the second (ii) from purified human monocytes (n= 753 individuals) collected during the CGP Project. SNPs associated with T1D are illustrated on the same scale (iii) to illustrate the co-localization of the eQTL and T1D association signals. Boundaries of intron 10 and intron 19 of CLEC16A contain the most associated SNPs for T1D and are contained within orange and black dotted lines, respectively. (C) DEXI expression by genotype at the T1D-associated SNP rs12708716 within the published GHS monocyte data set, where the minor (G) allele is protective, and (D) DEXI expression by genotype at the T1D-associated SNP rs725613 in the unpublished CGP monocyte data set.

Figure 1.

Expression of the DEXI gene, contained within the 16p13 region of T1D association (A) by microarray using Illumina probe set ILMN_1738866, is correlated with the genotype at SNPs within the CLEC16A gene in two independent data sets. The first data set (i) was generated from purified human monocytes (n= 1370 individuals) collected during GHS and the second (ii) from purified human monocytes (n= 753 individuals) collected during the CGP Project. SNPs associated with T1D are illustrated on the same scale (iii) to illustrate the co-localization of the eQTL and T1D association signals. Boundaries of intron 10 and intron 19 of CLEC16A contain the most associated SNPs for T1D and are contained within orange and black dotted lines, respectively. (C) DEXI expression by genotype at the T1D-associated SNP rs12708716 within the published GHS monocyte data set, where the minor (G) allele is protective, and (D) DEXI expression by genotype at the T1D-associated SNP rs725613 in the unpublished CGP monocyte data set.

Figure 2.

A long-range interaction was detected between the DEXI promotor region and intron 19 of CLEC16A in an EBV-transformed B cell line (yellow), the monocyte-like line THP-1 (blue) and the lung epithelial cell line A549 (red). Chromatin was cross-linked and digested by BglII and re-ligated. The interaction frequency between (A) the DEXI promotor fragment bait or (B) the 20 kb intron 19 region bait containing rs12708716 and distal candidate fragments was determined by qPCR and normalized to control template interactions generated using PCR-digested and ligated PCR products from genomic DNA. Error bars represent the standard error of three independent PCR reactions and peaks were confirmed by sequencing of the qPCR products. The two grey dashed lines represent the location of the DEXI promotor region and the region of intron 19 shown to interact with the DEXI promotor region.

Figure 2.

A long-range interaction was detected between the DEXI promotor region and intron 19 of CLEC16A in an EBV-transformed B cell line (yellow), the monocyte-like line THP-1 (blue) and the lung epithelial cell line A549 (red). Chromatin was cross-linked and digested by BglII and re-ligated. The interaction frequency between (A) the DEXI promotor fragment bait or (B) the 20 kb intron 19 region bait containing rs12708716 and distal candidate fragments was determined by qPCR and normalized to control template interactions generated using PCR-digested and ligated PCR products from genomic DNA. Error bars represent the standard error of three independent PCR reactions and peaks were confirmed by sequencing of the qPCR products. The two grey dashed lines represent the location of the DEXI promotor region and the region of intron 19 shown to interact with the DEXI promotor region.

Figure 3.

The interaction sites detected by 3C at the DEXI promotor region (A) and intron 19 of CLEC16A (B) are enriched for transcription-factor-binding sites (marked by grey and black horizontal lines) and enhancer-associated histone modifications (ENCODE project, displayed using the UCSC Genome browser). Intron 19 of CLEC16A also contains RNA polymerase II binding peaks. The 3C interaction fragment, between two BglII restriction enzyme digestion sites, is highlighted with a yellow line.

Figure 4.

The eQTL and 3C data allow a model to be proposed in which a DNA loop is formed between the DEXI promotor region and intron 19 of CLEC16A. In this model, the T1D-associated SNPs within the loop affect gene transcription by influencing the binding of transcription factors and other proteins at or near the interacting site in intron 19. A more efficient transcription complex is formed in the presence of protective T1D alleles at 16p13, allowing more efficient DEXI transcription. It is also possible that a converse model is true, with protective alleles being associated with reduced binding of suppressor/silencer proteins.