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A7760 - An IFITM3 SNP Associated with Influenza Severity Found to Affect Chromatin Interactions
Author Block: E. K. Allen1, D. J. Downes2, J. R. Hughes2, P. G. Thomas1; 1Immunology, St. Jude Children's Research Hospital, Memphis, TN, United States, 2Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
Rationale: IFITM3, a known antiviral protein, has been shown to both restrict viral replication in epithelial cells as well as promote survival of tissue resident CD8+ T-cells in mouse studies. We previously identified a SNP (rs34481144) in the promoter region of IFITM3 as being associated with severe influenza illness in three independent cohorts. We further characterized the SNP as shifting transcription factor binding and being correlated with both IFITM3 protein levels in CD8 T-cells and the number of CD8 T-cells at site of infection in individuals with influenza infection. Based on our findings that rs34481144 is in the promoter of IFITM3 and affects binding of transcription factors, we sought to determine how the genotype would affect chromatin structure and function in CD8 T-cells.
Methods: In order to look at chromatin structure differences, we used the Low Input Capture-C method in CD8 T-cells from patients of different homozygous rs34481144 genotypes. We enriched CD8 T-cells from six healthy donors, three of which had the A/A (risk) genotype and three of which had the G/G (protective) genotype. We used Capture-C combining the 3C method for chromatin confirmation and targeted sequencing to capture specific regions of interest to determine if chromatin interactions differed between genotypes. Using CapSequm, we designed 33 probes covering 23 genes in the region surrounding rs34481144 for the sequence capture.
Results: The final Capture-C sample was sequenced on the Illumina HiSeq 2500 and analyzed for chromatin interactions using the NG-Capture-C analysis pipeline. We successfully performed Capture-C on CD8 T-cells from six healthy donors based on their rs34481144 genotype. With 96% capture efficiency, we had very high quality sequence data from these individuals to look for differences in chromatin structure. From our Capture-C data, we found evidence of differential chromatin interactions between genotypes with the protective allele showing preference of IFITM3 to interact with the region at IFITM1 and IFITM2 and the risk allele showing preference to an intergenic region upstream.
Conclusion: Our data provide evidence for differential chromatin conformation in CD8 T-cells based on rs34481144 genotype. We plan to repeat the Capture-C in more individuals of distinct ancestral backgrounds as well as look at differences in chromatin accessibility by ATAC-seq and protein binding with ChIP-seq between rs34481144 genotypes to fully understand the regulation in this region and how it may affect host response to influenza infection.