Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis
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,...
| Main Authors: | , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Wiley
2021
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| _version_ | 1826307853686669312 |
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| author | Vecellio, M Chen, L Cohen, CJ Cortes, A Li, Y Bonham, S Selmi, C Brown, MA Fischer, R Knight, JC Wordsworth, BP |
| author_facet | Vecellio, M Chen, L Cohen, CJ Cortes, A Li, Y Bonham, S Selmi, C Brown, MA Fischer, R Knight, JC Wordsworth, BP |
| author_sort | Vecellio, M |
| collection | OXFORD |
| description | 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 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.
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.
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| first_indexed | 2024-03-07T07:09:17Z |
| format | Journal article |
| id | oxford-uuid:7a11dd7c-1926-4b9e-b424-549e38be6deb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:09:17Z |
| publishDate | 2021 |
| publisher | Wiley |
| record_format | dspace |
| spelling | oxford-uuid:7a11dd7c-1926-4b9e-b424-549e38be6deb2022-06-07T13:19:18ZFunctional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitisJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:7a11dd7c-1926-4b9e-b424-549e38be6debEnglishSymplectic ElementsWiley2021Vecellio, MChen, LCohen, CJCortes, ALi, YBonham, SSelmi, CBrown, MAFischer, RKnight, JCWordsworth, BPObjective 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 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. 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. |
| spellingShingle | Vecellio, M Chen, L Cohen, CJ Cortes, A Li, Y Bonham, S Selmi, C Brown, MA Fischer, R Knight, JC Wordsworth, BP Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis |
| title | Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis |
| title_full | Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis |
| title_fullStr | Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis |
| title_full_unstemmed | Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis |
| title_short | Functional genomic analysis of a RUNX3 polymorphism associated with ankylosing spondylitis |
| title_sort | functional genomic analysis of a runx3 polymorphism associated with ankylosing spondylitis |
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