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

Carla J Cohen; Connor Davidson; Carlo Selmi; Paul Bowness; Julian C Knight; B Paul Wordsworth; Matteo Vecellio

doi:10.3389/fgene.2021.741867

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

Front Genet. 2022 Jan 7:12:741867. doi: 10.3389/fgene.2021.741867. eCollection 2021.

Authors

Carla J Cohen^{1

2}, Connor Davidson^{1

2

3}, Carlo Selmi⁴, Paul Bowness^{1

2}, Julian C Knight³, B Paul Wordsworth^{1

2}, Matteo Vecellio^{1

2

4}

Affiliations

¹ Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom.
² National Institute for Health Research Oxford Comprehensive Biomedical Research Centre, Botnar Research Centre, Nuffield Orthopaedic Centre, Oxford, United Kingdom.
³ Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
⁴ Division of Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center - IRCCS, Rozzano, Italy.

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.

Keywords: RUNX3; ankylosing spondylitis; c-Myc; chromosome conformation capture (3C); single nucleotide polymorphism (SNP).