Functional Genomic Analysis of a RUNX3 Polymorphism Associated With Ankylosing Spondylitis

Abstract

Objective. To 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).Methods. Using 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 pulleddown 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.Results. In 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).Conclusion. These 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.

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

“…We accept that recent findings highlight the fact that contact frequencies from 3C assays sometimes do not correspond to 3D proximity (Williamson et al, 2014), but taken together with the functional data presented here and other recently published findings (Vecellio et al, 2016;Vecellio et al, 2018;Vecellio et al, 2021) we are confident in our results. However, we are aware that other higher throughput techniques have been developed (eg.…”

“…It also influences many other cells, including helper T-cells, innate lymphoid, tissue resident, mucosa and gut cells (Ebihara et al, 2015;Behr et al, 2018). We have recently demonstrated that ASassociated non-coding single nucleotide polymorphisms (SNPs) in an enhancer-like region upstream of RUNX3 affect the binding of different factors: in particular the repressive nucleosome remodelling and deacetylase (NuRD) complex binds preferentially to the risk allele, while conversely interferon regulatory factor (IRF) five to the protective allele (Vecellio et al, 2021). However, the functional effects of these changes on gene transcription are still to be precisely determined.…”

Disruption of c-MYC binding and chromosomal looping involving genetic variants associated with ankylosing spondylitis upstream of RUNX3 promoter

“…Here, we have demonstrated physical interaction between the distal promoter of RUNX3 and rs4648889 SNP, which we have previously functionally characterized by our group. (17, 35) Conversely, a very low interaction was observed with rs6600247 suggesting no evident functional role for this SNP in chromosome looping in this particular cellular context.…”

“…(14) It also influences many other cells, including helper T-cells, innate lymphoid, tissue resident, mucosa and gut cells (15, 16). We have recently demonstrated that AS-associated non-coding single nucleotide polymorphisms (SNPs) in an enhancer-like region upstream of RUNX3 affect the binding of different factors: in particular the repressive nucleosome remodelling and deacetylase (NuRD) complex binds preferentially to the risk allele, while conversely interferon regulatory factor (IRF) 5 to the protective allele (17). However, the functional effects of these changes on gene transcription are still to be precisely determined.…”

Perspectives on the Genetic Associations of Ankylosing Spondylitis

“…In our lab, we have so far demonstrated that the RUNX3 ASassociated SNP rs4648889 (above) mediates differential allelic binding of two regulatory factors/complexes to a putative enhancer in the region upstream of the promoter: (1) the transcription factor interferon regulatory factor (IRF) 5, which binds preferentially to the AS-protective "G" allele; and (2) components of the nucleosome remodeling and deacetylase (NuRD) complex (one of the four major ATP-dependent chromatin remodeling complexes that function as transcriptional repressors) bind preferentially instead to the AS-risk "A" allele at rs4648889 (64). Further work is necessary to confirm the functional consequences of this SNP on gene expression and the network of genes involved but preliminary experiments suggest that IRF5 knockdown in CD8+ T-cells reduces the expression of interferon gamma.…”