Furthermore, we detected an conversation between the promoters of and that was not revealed in the Region Capture experiment, as promoters were excluded from the Region Capture experiment (Fig.?1l). Importantly, we sought validation of CHi-C results by 3C-quantitative real-time polymerase chain reaction (qPCR). the next important step for the translation of genetic findings into biologically meaningful mechanisms of disease and potential therapeutic targets. GB1107 Using novel chromatin conversation detection techniques and allele specific assays in T and B cell lines, we provide compelling evidence that redefines causal genes at the 6q23 locus, one of the most important loci that confers autoimmunity risk. Results Although the function of disease-associated non-coding single nucleotide polymorphisms (SNPs) at 6q23 is usually unknown, the association is generally assigned to gene, the closest most plausible causal gene within the locus, with impartial variants within the gene associated with different diseases. There are three distinct linkage disequilibrium (LD) blocks independently associated with a range of autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), celiac disease (CeD), type 1 diabetes (T1D), inflammatory bowel disease (IBD), psoriasis (Ps) and psoriatic arthritis (PsA) [22C29]. One region, containing SNPs associated with RA, SLE, CeD, IBD and T1D, tagged by the rs6920220 SNP, lies a considerable distance ( 181?kb) from the gene and its functional role has, so far, been underexplored (Fig.?1g). The second, impartial association signal, tagged by rs7752903, and predisposing to RA, SLE and CeD, spans around 100?kb and includes the gene (Fig.?1h). There is evidence that a TT? ?A polymorphism located within this LD block, 42?kb downstream of promoter [9, 30, 31]. An additional association signal, tagged by rs610604, confers risk to Ps and PsA (Fig.?1i). Open in a separate windows Fig. 1 Long-range interactions in the 6q23 locus. Genomic co-ordinates are shown along the of each and are labelled aCn. a HindIII restriction fragments. bCe Regions targeted and restriction fragments included in the Region (b, c) and Promoter (d, e) Capture experiments. f GENCODE V17 genes. gCi 1000 Genomes SNPs in LD (r2??0.8) with the index SNPs rs6920220, associated with RA, SLE, celiac disease, T1D and IBD (g), rs7752903, associated with RA, SLE and celiac disease (h) and rs610604, associated with Ps and PsA (i). j Topologically associated domains (TADs) in GM12878 cells [20]. kCn Significant interactions identified in the Region and Promoter capture experiments in GM12878 (k, l) and Jurkat (m, n) cells. The indicates the position of the rs6927172 SNP The aim of the current work was to identify causal disease genes and refine the likely causal SNPs at the autoimmunity locus 6q23 by studying long-range chromatin interactions using CHi-C, to validate findings using genotype specific 3C and augment the evidence further with cell-type and genotype specific expression quantitative trait loci (eQTL) and chromatin immunoprecipitation (ChIP) analysis. Here, we report a new causal candidate disease gene within the 6q23 region, promoter, resulting in increased expression of the gene. Results 6q23 variants interact with several genes, including and gene. The Region Capture experiments targeting both the LD block containing RA (rs7752903) and Ps/PsA (rs610604) associated variants, and spanning the gene along with its upstream and downstream ATP7B regions (Fig.?1h and i), showed interactions with a region proximal to the rs6920220 LD block, encompassing the lncRNAs RP11-95M15.2 (a pseudogene) and RP11-356I2.1, the miRNA “type”:”entrez-nucleotide”,”attrs”:”text”:”AL357060.1″,”term_id”:”8247186″,”term_text”:”AL357060.1″AL357060.1 and also an upstream region containing non-coding RNAs (Y_RNA and RP11-356I2.2) (Fig.?1k). Finally, the Region Capture experiment detected an interaction involving and a region containing the lncRNAs RP11-10J5.1 and RP11-240M16.1 approximately 50?kb downstream of the gene, which in turn, also interacts with the intergenic rs6920220-tagged LD block. Interestingly, this region, downstream of gene (Fig.?1k). These interactions were independently validated in the GB1107 second, separate Promoter Capture experiment (Fig.?1d, e, l and n). Furthermore, we detected an interaction between the promoters of and that was not revealed in the Region Capture experiment, as promoters were excluded from the Region Capture experiment (Fig.?1l). Importantly, we sought validation of CHi-C results by 3C-quantitative real-time polymerase chain reaction (qPCR). Higher interaction frequencies were confirmed for all interrogated regions, compared to adjacent noninteracting regions (Fig.?2). Open in a separate window Fig. 2 Validation of CHi-C results by 3C-qPCR in GM12878 and Jurkat cell lines. The show the relative interaction frequency of (a) the 6q23 intergenic disease SNPs tagged by rs6920220, (b) the gene and (c) the gene with their respective targets (below each show the approximate location of the primers for the anchor, negative control (C-) and target () regions. indicate standard deviation GB1107 of three biological replicates; * indicates t-test value 0.05 To validate our analysis method, we.