Modelling human pancreatic beta cell enhancer function in diabetes

点击翻译按钮获取中文摘要

Diabetes mellitus is a complex metabolic disorder characterised by chronic hyperglycemia that affects over 400 million individuals worldwide. With over 1.6 million deaths reported in 2015, diabetes represents an increasing global health threat that is expected to become the 7th leading cause of mortality by 2030. This increase has largely been associated with type II diabetes (T2D), which accounts for 90% of cases, and has been tied to changes in human lifestyle and behaviour, and increasing rates of obesity. Although environmental and behavioural factors, such as a sedentary lifestyle and poor diet, are known risk factors of T2D, contributing to the development of chronic insulin resistance, recent studies have highlighted the contribution of genetic factors to diabetes disease risk, and a strong heritable component to T2D has been established.Genome wide association studies (GWAS) have identified more than 100 T2D-associated loci, the majority of which impact pancreatic Beta cell function and insulin secretion. Disease and trait-associated variants are concentrated in non-coding regions of the genome, which can be characterised by cis-acting regulatory elements such as enhancers, which play a key role in cell type-specific, signal-responsive transcription. Enhancers act as scaffolds for transcription factors, driving gene expression through physical interactions with target promoters, irrespective of distance or orientation, and regulatory mutations within these elements have been associated with rare, monogenic forms of diabetes, such as pancreas agenesis. The principles that determine whether or not an enhancer sequence variant is 'damaging', however, are not well understood. Enhancers are remarkably diverse and demonstrate stark differences in sequence composition and transcription factor binding, as well as evolutionary constraints in different tissues. As the majority of regulatory variants have been shown to have little phenotypic effect, determining which regulatory variants contribute to disease risk presents a significant challenge to current research.This project aims to investigate the molecular consequences of enhancer mutations in pancreatic Beta cells, to develop a better understanding of enhancer biology and the contribution of regulatory mutations to diabetes disease risk. Genome engineering approaches, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, provide a means through which specific genomic sequences can be targeted and modified. Recent advances in single cell RNA sequencing (scRNA-seq) have led to the development of single cell CRISPR screens that can detect alterations of enhancer activity, providing a new, powerful tool for investigating the phenotypic impact of regulatory mutations in their native genomic and cell-type specific context. Using a combination of these methods, a single cell genomics strategy will be used to investigate the phenotypic impact of regulatory mutations on human pancreatic Beta cells. Integrative analysis will be used to assess key features of impactful enhancer mutations in human pancreatic islets, providing an insight into the underlying principles of enhancer biology that determine which enhancer variants elicit strong cellular phenotypes. Overall, this project will provide an insight into the molecular consequences of disease-associated regulatory mutations, and the contribution of enhancer mutations to diabetes disease risk and pathogenesis.