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

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.

Chromosome conformation capture approaches to investigate 3D genome architecture in Ankylosing Spondylitis

“…The role of specific GWAS hits can be elucidated via 3D genome analysis, thus clarifying which genes are influenced by which particular SNP through a spatial connection ( Li et al, 2020 ). Our group has recently demonstrated the presence of a chromatin loop between the AS-associated SNP rs4648889 and the distal promoter of the RUNX3 gene, confirming together with other functional experiments previously reported the primacy of this genetic variant in the association with RUNX3 in AS ( Cohen et al, 2021 ). The complexity of the RUNX3 locus is also confirmed by Capture-C experiments showing multiple interactions among different SNPs and the RUNX3 promoter (personal communication).…”

p53 Deficiency-Dependent Oncogenicity of Runx3

“…Retroviral insertional mutagenesis screens have shown that all three Runx genes act as collaborating oncogenes in Myc-driven lymphoma mouse models [ 13 , 68 , 69 , 70 , 71 ]. These findings were supported by results showing that RUNX and MYC expression are positively correlated in various biological activities [ 16 , 17 , 72 , 73 , 74 ]. In T-cell acute lymphoblastic lymphoma cells, RUNX3 and RUNX1 bind the +1.43 Mb MYC enhancer N-Me and upregulate MYC expression [ 16 ].…”

Comprehensive epigenomic profiling reveals the extent of disease-specific chromatin states and informs target discovery in ankylosing spondylitis

“… 53 ETS1 regulates the expression of IL-7R 54 (encoding the IL-7Rα subunit), which is also associated with AS, 20 and RUNX3 , which encodes Runt-related TF 3, a TF involved in T cell function with known AS-associated functional variants. 15 , 16 , 17 , 18 ETS1 , ETS2 , and IL7R are in the Pi network output ( Figure 6 D), indicating that they form part of an important functional pathway with strong possibility for therapeutic intervention.…”

“… 9 , 13 , 14 However, most AS associations involve non-coding SNPs, which may be regulatory in nature and act in a cell-specific manner to modulate a variety of epigenetic, transcriptional, and post-transcriptional mechanisms. 15 , 16 , 17 , 18 Recently we demonstrated how AS-associated SNPs at RUNX3 modulate the binding of transcription factors (TFs) and regulatory complexes in T cells and monocytes, 15 , 16 , 17 , 18 but for other associated loci, the causal genes and pathways remain largely unresolved. 19 The expression and co-ordination of regulatory mechanisms for genes involved in the disease pathophysiology are likely to be cell type specific.…”