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Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter.

ABSTRACT: Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10^-15) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14⁺ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk "C" allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

SUBMITTER: Cohen CJ

PROVIDER: S-EPMC8782160 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveTo investigate the functional consequences of the single-nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter associated with susceptibility to ankylosing spondylitis (AS).MethodsUsing nuclear extracts from Jurkat cells and primary human CD8+ T cells, the effects of rs4648889 on allele-specific transcription factor (TF) binding were investigated by DNA pull-down assay and quantitative mass spectrometry (qMS), with validation by electrophoretic mobility shift assay (EMSA), Western blotting of the pulled-down eluates, and chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis. Further functional effects were tested by small interfering RNA knockdown of the gene for interferon regulatory factor 5 (IRF5), followed by reverse transcription-qPCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) to measure the levels of IFNγ messenger RNA (mRNA) and protein, respectively.ResultsIn nuclear extracts from CD8+ T cells, results of qMS showed that relative TF binding to the AS-risk A allele of rs4648889 was increased 3.7-fold (P < 0.03) for Ikaros family zinc-finger protein 3 (IKZF3; Aiolos) and components of the NuRD complex, including chromodomain helicase DNA binding protein 4 (CHD4) (3.6-fold increase; P < 0.05) and retinoblastoma binding protein 4 (RBBP4) (4.1-fold increase; P < 0.03). In contrast, IRF5 bound significantly more to the AS-protective G allele compared to the AS-risk A allele (fold change 8.2; P = 0.003). Validation with Western blotting, EMSA, and ChIP-qPCR confirmed the differential allelic binding of IKZF3, CHD4, RBBP4, and IRF5. Silencing of IRF5 in CD8+ T cells increased the levels of IFNγ mRNA as measured by RT-qPCR (P = 0.03) and IFNγ protein as measured by ELISA (P = 0.02).ConclusionThese findings suggest that the association of rs4648889 with AS reflects allele-specific binding of this enhancer-like region to certain TFs, including IRF5, IKZF3, and members of the NuRD complex. IRF5 may have crucial influences on the functions of CD8+ lymphocytes, a finding that could reveal new therapeutic targets for the management of AS.

Project description:We have compared synovial biopsies from ankylosing spondylitis and undifferentiated spondylitis patients with healthy controls and osteoarthritis patients Objective: In spondylarthropies, whole-genome gene expression profiling studies have been limited to peripheral blood to date. By undertaking a study in knee synovial biopsies from spondylarthropy (SpA) and ankylosing spondylitis (AS) patients we aimed to identified joint-specific candidate genes and pathways. These pathways may mediate systemic inflammation driven joint damaging processes and more specifically, the osteoproliferation that is characteristic of these conditions. Methods: RNA was extracted from six seronegative SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: 416 differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, osteoblast activity, B-cell associated, matrix catabolic, and metabolic pathways. The inflammatory mediator, MMP3, was strongly upregulated in AS/SpA samples and the Wnt pathway inhibitors DKK3 and Kremen1 were downregulated. Conclusion: Pathways mediating both systemic inflammation as well as local tissue changes were identified. This suggests initial systemic inflammation in spondylarthropies transfers to and persists in the local joint environment, subsequently mediating changes in genes directly involved in the destructive tissue remodelling. Fifteen knee synovial biopsy tissue samples consisting of six seronegative spondyloarthropy (SpA), two ankylosing spondylitis (AS), three osteoarthritis (OA) and four normal control biopsies were obtained from the Synovial Tissue Bank at the Repatriation General Hospital in Adelaide, South Australia with the appropriate ethical approval (Supplementary Table 1). All patients provided informed written consent.

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Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter.

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