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.
Highlights
BackgroundAnkylosing Spondylitis (AS) is a form of inflammatory spondyloarthritis predominantly affecting the axial skeleton, which is characterised pathologically by enthesitis (Bridgewood et al, 2020)
single nucleotide polymorphisms (SNPs) rs6600247 is situated within a region of open chromatin, defined by a peak for dnase I hypersensitvity (DHS - indicative of regions of open chromatin) (Figure 1B), and a peak of H3K4Me1 histone modification
This sequence binds the transcription factor c-MYC (ENCODE Factorbook (Figure 1B). These data suggest an enhancer-type element surrounding rs6600247, so we sought to determine a regulatory role of this SNP
Summary
BackgroundAnkylosing Spondylitis (AS) is a form of inflammatory spondyloarthritis predominantly affecting the axial skeleton, which is characterised pathologically by enthesitis (Bridgewood et al, 2020). AS was one of the first complex diseases in which a specific genetic effect was identified when its strong association with the major histocompatibility complex (MHC) immune response gene HLA-B27 was described nearly 50 years ago (Brewerton et al, 1973) (Schlosstein et al, 1973). 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 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)
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