Alzheimer’s disease (AD) is characterised by distinctive molecular changes across multiple cell types in the human brain. Epigenome-wide association studies (EWAS) have identified widespread alterations in epigenetic modifications associated with AD pathology, but a major challenge remains: which of these changes have functional consequences for gene regulation and disease biology. This question is particularly important in the brain, where different cell types have highly distinct regulatory landscapes. Using fluorescence-activated nuclei sorting (FANS), we have generated cell-type-specific epigenomic datasets from human post-mortem brain, including DNA methylation, chromatin accessibility (ATAC-seq) and H3K27 acetylation (H3K27ac), a marker of active regulatory elements (Ref. 1).
These unique datasets provide an opportunity to move beyond association and identify disease-associated epigenetic changes occurring within functional regulatory elements in specific brain cell types. The central question of this PhD is to identify AD-associated regions with regulatory potential, and to functionally characterise how they influence gene regulation and cellular phenotype. The project will address this through three complementary objectives. First, the student will integrate cell-type-specific DNA methylation, ATAC-seq and H3K27ac data to identify and prioritise candidate regulatory loci from our AD EWAS (Exeter).
These data will be combined with available gene expression, genetic association, and regulatory annotations to identify disease-associated epigenetic changes occurring within active promoters and enhancers and establish their likely target genes. This multi-omic approach will move beyond conventional annotation of EWAS signals based simply on proximity to the nearest gene and instead prioritise loci with strong evidence for a functional regulatory role. Second, selected regulatory loci will be functionally interrogated using targeted CRISPR epigenome editing. Catalytically inactive Cas9 (dCas9) fused to DNA methylation, demethylation, or chromatin remodelling enzymes will be used to induce or reverse disease-associated epigenetic states in relevant cell lines and human induced pluripotent stem cell (hiPSC)-derived neural cells (cortical neurons/microglia).
These novel methods are currently being optimised by the supervisory team funded by an FGx innovation and collaboration fund award (PI Bello, Milner Institute). Initial studies will focus on microglia, where we have identified prominent AD-associated DNA methylation differences and the possess relevant expertise (Washer), but still allowing the student to investigate neuronal or other glial mechanisms emerging from the computational analyses. Third, the student will determine the molecular and cellular consequences of these perturbations. Targeted assays will confirm epigenetic editing (e.g. bisulfite pyrosequencing), followed by analysis of gene expression and relevant cellular phenotypes.
For the strongest candidates, transcriptomic, methylomic and/or chromatin-accessibility profiling will establish how perturbation alters regulatory networks and cellular states. This interdisciplinary project combines human disease epigenomics, computational biology, stem-cell models and CRISPR-based functional genomics. The student will develop expertise spanning multi omic data integration, reproducible bioinformatics, experimental epigenomics, cell culture, epigenome editing and functional assays. Importantly, the initial analyses will allow the student to shape which loci, cell types and biological pathways are pursued experimentally.
The project is supported by extensive existing cell-type-specific datasets, established FANS and epigenomic pipelines (Dempster & Mill, Exeter), access to hiPSC-derived models of neurodegeneration (Washer (Oxford) & Bello (Cambridge)), omics (Dempster & Mill, Exeter), and expertise in targeted DNA methylation editing (Exeter, Oxford and Cambridge)
