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

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 which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP ( = 2.6 × 10) lies ∼13kb upstream of the promoter adjacent to a c-MYC TF binding-site. The effect of 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). Interrogation of ENCODE published data showed open chromatin in the region overlapping in primary human CD14 monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning 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 ( = 3; < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the promoter and the previously characterised AS-associated SNP . The lead SNP located in an enhancer-like region upstream of the promoter, modulates c-MYC binding. However, the region encompassing has rather limited physical interaction with the promoter of . In contrast a clear chromatin looping event between the region encompassing and the promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the promoter and the involvement of transcriptional regulation in AS.