Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

<h4>Objectives</h4>To investigate the functional consequences of the single nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter associated with ankylosing spondylitis (AS).<h4>Methods</h4>The effects of rs4648889 on allele-specific transcription factor (TF) binding were investigated by DNA pull-down and quantitative mass spectrometry (qMS) with validation by electrophoretic mobility gel shift assays (EMSA), Western blot (WB) analysis of the pulled-down eluates, and chromatin immuno-precipitation (ChIP)-qPCR. Further functional effects were tested by siRNA knockdown of IRF5 followed by qRT-PCR and ELISA to measure mRNA and protein levels of IFNγ.<h4>Results</h4>Using nuclear extracts from primary human CD8+ T-cells assessed by qMS, relative TF binding to the AS-risk "A" allele of rs4648889 was increased (3.7-fold, p<0.03) for IKZF3 (aiolos) and components of the NUcleosome Remodeling Deacetylase (NuRD) complex, including Chromodomain-Helicase-DNA-binding protein (CHD) 4 (3.6-fold, p<0.05) and Retinoblastoma-Binding Protein (RBBP) 4 (4.1-fold, p<0.02). In contrast, interferon regulatory factor (IRF) 5 bound significantly less to the A allele (8.2-fold, p=0.003). Validation with WB, EMSA and ChIP-qPCR confirm differential allelic binding for IKZF3, CHD4, RBBP4 and IRF5. Silencing of IRF5 in CD8+ T-cells increased IFNγ mRNA (measured by RT-qPCR (p=0.03) and protein by (ELISA p=0.02).<h4>Conclusions</h4>The 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 CD8+ lymphocyte function that could reveal new therapeutic targets in AS.

Original publication

DOI

10.1002/art.41628

Type

Journal article

Journal

Arthritis & rheumatology (Hoboken, N.J.)

Publication Date

26/12/2020

Addresses

Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK.