Modelling human pancreatic beta cell enhancer function in diabetes

糖尿病是一种以慢性高血糖为特征的复杂代谢紊乱，影响全球超过4亿人。2015年报告的死亡人数超过160万，糖尿病是一种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。这种增加在很大程度上与II型糖尿病（T2 D）有关，占病例的90%，并且与人类生活方式和行为的变化以及肥胖率的增加有关。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T2 D的已知危险因素，有助于慢性胰岛素抵抗的发展，但最近的研究已经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T2 D的强遗传成分。全基因组关联研究（GWAS）已经鉴定了超过100个T2 D相关基因座，其中大部分影响胰腺β细胞功能和胰岛素分泌。疾病和性状相关变异集中在基因组的非编码区，其特征在于顺式作用调控元件，如增强子，其在细胞类型特异性的信号响应转录中起关键作用。增强子作为转录因子的支架，通过与靶启动子的物理相互作用驱动基因表达，而不管距离或方向如何，并且这些元件内的调控突变与罕见的单基因型糖尿病有关，例如胰腺发育不全。然而，确定增强子序列变体是否是“破坏性的”的原理还没有很好地理解。增强子是非常多样的，并在序列组成和转录因子结合，以及在不同组织中的进化限制表现出明显的差异。由于大多数调控变异已被证明几乎没有表型效应，确定哪些调控变异有助于疾病风险是当前研究的一个重大挑战。本项目旨在研究胰腺β细胞中增强子突变的分子后果，以更好地了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，如成簇的规则间隔短回文重复序列（CRISPR）/Cas9系统，提供了一种可以靶向和修饰特定基因组序列的手段。单细胞RNA测序（scRNA-seq）的最新进展已经导致单细胞CRISPR筛选的发展，可以检测增强子活性的改变，为研究调控突变在其天然基因组和细胞类型特异性背景下的表型影响提供了一种新的强大工具。使用这些方法的组合，将使用单细胞基因组学策略来研究调节突变对人胰腺β细胞的表型影响。整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，这些原理确定哪些增强子变体引起强细胞表型。总的来说，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.