The identification of genetic and epigenetic changes that contribute to T1D (Type 1 Diabetes) by ATAC-seq (Assay of Transposase Accessible Chromatin with high throughput sequencing)

The exact cause for T1D is unknown, but the contribution of genetic and environmental factors, leading to a breakdown in immune tolerance has been proposed. Genetic data strongly link the breakdown in tolerance to alterations in the transcriptional program in CD4⁺ T cells. Various environmental factors associated with T1D have also been proposed, but how these causes disease is not well characterised. Emerging studies suggest that epigenetic changes are a likely route for environmental factors to alter the immune system.  ATAC-seq is a new genome-wide method to simultaneously probe chromatin structure (accessibility and nucleosome positioning) and importantly, map TF (transcription factor) binding at high resolution. We will use ATAC-seq to compare the DNA accessibility and TF binding profiles in Treg and Tconv cells from individuals with established T1D and healthy controls. This will allow identification of changes in chromatin structure and differential TF binding in T1D samples relative to healthy controls to fine map changes that are associated with the altered transcriptional program in T1D. We have optimised thawing of frozen biobanked PBMCs and established the protocol for isolating sufficient numbers of highly pure and viable Tregs and Tconv cells for ATAC-Seq from thawed PBMCs. Having performed a pilot ATAC-seq experiment to determine the optimal nuclei tagmentation time, ATAC-seq will be performed on fresh and frozen healthy T cells (Tregs and Tconv) cells, followed by age-matched T1D samples, and high resolution mapping of accessible chromatin regions and any differences between cell types and between healthy and t1D samples will be analysed. When combined with mapping of genetic variation linked to T1D, this may shed insight into which regions of the genome are modifiable and whether these regions are differentially modified when there is genetic variation present.