Modelling human pancreatic beta cell enhancer function in diabetes

糖尿病是一种复杂的代谢紊乱，以慢性高血糖为特征，全世界有超过4亿人受到影响。据报道，2015年有超过160万人死于糖尿病，糖尿病是一个日益严重的全球健康威胁，预计到2030年将成为第七大死亡原因。这一增长在很大程度上与占病例90%的II型糖尿病(T2D)有关，并与人类生活方式和行为的变化以及肥胖率的上升有关。虽然环境和行为因素，如久坐不动的生活方式和不良饮食，是T2D的已知危险因素，导致慢性胰岛素抵抗的发生，但最近的研究强调了遗传因素对糖尿病疾病风险的贡献，并且已经建立了T2D的一个强大的可遗传成分。基因组广泛关联研究(GWAS)已经确定了100多个T2D相关基因，其中大多数影响胰腺β细胞功能和胰岛素分泌。疾病和性状相关的变异体集中在基因组的非编码区，这些非编码区可以由顺式作用的调控元件(如增强子)来表征，这些元件在细胞类型特定的信号响应转录中发挥关键作用。增强子作为转录因子的支架，通过与目标启动子的物理相互作用来驱动基因表达，而不考虑距离或方向，这些元件中的调节性突变与罕见的单基因形式的糖尿病有关，如胰腺发育不全。然而，确定增强子序列变体是否具有破坏性的原理还没有被很好地理解。增强子有很大的多样性，在序列组成、转录因子结合以及不同组织中的进化限制方面表现出明显的差异。由于大多数调控变异体几乎没有表型效应，确定哪些调控变异体对疾病风险有重大影响是当前研究的一个重大挑战。本项目旨在研究胰腺β细胞增强子突变的分子后果，以更好地理解增强子生物学和调控突变对糖尿病疾病风险的贡献。基因组工程方法，如簇状规则间隔短回文重复(CRISPR)/Cas9系统，提供了一种特定基因组序列可以被靶向和修饰的手段。单细胞RNA测序(scRNA-seq)的最新进展导致了单细胞CRISPR筛选的发展，可以检测增强子活性的变化，为研究调节突变在其天然基因组和细胞类型特定背景下的表型影响提供了一个新的、强大的工具。结合使用这些方法，将使用单细胞基因组学策略来研究调节性突变对人胰岛β细胞的表型影响。综合分析将用于评估人类胰岛中有影响力的增强子突变的关键特征，从而深入了解增强子生物学的基本原理，这些原理决定了哪些增强子变异会引起强烈的细胞表型。总体而言，该项目将深入了解疾病相关调控突变的分子后果，以及增强子突变对糖尿病疾病风险和发病机制的贡献。

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.