Pos0031 functional genomics investigation of the ankylosing spondylitis associated locus runx3

BackgroundAnkylosing Spondylitis (AS), is a highly heritable disease with >100 genomic loci incriminated. Among these, RUNX3, a transcription factor (TF) involved in diverse immunological processes, is robustly (10−15) associated. [1] We have extensively investigated the single nucleotide polymorphism (SNP) rs4648889 located in a 2kb regulatory locus upstream of the RUNX3 promoter. We have demonstrated that the association between AS and this SNP can be explained by allele-specific effects on TF recruitment (including IRF4, IRF5 and the NuRD complex) that alter gene expression, specifically in CD8+ T-cells, and having a crucial role in CD8+ T-cells function. [2, 3]Further, we have recently shown a clear chromatin looping event between the region encompassing SNP rs4648889 and the RUNX3 promoter confirming the functional role of this genetic variant. [4]ObjectivesThe purpose of this work is: (1) to better characterise the chromatin looping landscape of the whole AS-associated RUNX3 genomic locus and (2) to determine the single-cell expression of the RUNX3-related genes identified previously [2, 3] in CD8+ T-cells lymphocytes.Methods(1) Chromosome conformation capture (3C) technique followed by qPCR was performed to define the chromatin looping events at the RUNX3 genomic locus in relevant cell lines, including Jurkat T-cells and U937 monocytes-like cells. (2) 10X Chromium single-cell (sc) sequencing was performed to define RUNX3 and RUNX3-related genes expression profile in CD8+ T cells obtained from AS cases.Results(1) In addition to the recent results published, [4] 3C-qPCR experiments revealed a high interaction frequency between the distal promoter of RUNX3 and an intronic region (called Int2), overlapping open chromatin and TF binding sites. This was highly reproducible in both cell lines analysed. (2) Four different clusters were identified in CD8+ T-cells obtained from AS peripheral blood via 10x sc-seq based on the expression of RUNX3. Five other genes (TBX21, EOMES, CHD4, IRF5 and IKZF3) previously identified [2] were considered: cluster 1 showed a strong co-expression for RUNX3, IKZF3, CHD4 and EOMES. Further analysis is required to better characterize this subpopulation.ConclusionThe AS-associated RUNX3 genomic locus has a plausible functional role in AS, probably by regulating gene transcription and DNA looping. These observations are critically important in defining dysregulated pathways and potential therapeutic drug targets.References[1]IGAS et al. Nat Genet. 2013 Jul;45(7):730-8.[2]Vecellio M. et al, Ann Rheum Dis. 2016 Aug;75(8):1534-40.[3]Vecellio M. et al, Arthritis Rheumatol 2020. doi: 10.1002/art.41628.[4]Cohen CJ. et al, Front Genet 2022 doi.org/10.3389/fgene.2021.741867.Disclosure of InterestsCarla Cohen: None declared, Davide Simone: None declared, Carlo Selmi Speakers bureau: Speakers fee (AbbVie, Amgen, Alfa-Wassermann, Biogen, Celgene, Eli-Lilly, Gilead, Janssen, MSD, Novartis, Pfizer, Sanofi-Genzyme), Consultant of: Consulting (AbbVie, Amgen, Alfa-Wassermann, Biogen, Celgene, Eli-Lilly, Gilead, Janssen, MSD, Novartis, Pfizer, Sanofi-Genzyme), Grant/research support from: Research support (AbbVie, Amgen, Janssen, Pfizer), aul Bowness Grant/research support from: Regeneron, Celgene/BMS and GSK, Paul Bryan Wordsworth: None declared, Matteo Vecellio: None declared