Functional Mapping of Enhancer Conservation Between Species to Enable Mechanistic Insights into Polygenic Disease

物种间增强子保护的功能图谱，以实现对多基因疾病的机制洞察

基本信息

批准号：
10294279
负责人：
Ryan Tewhey
金额：
$ 51.92万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10294279
关键词：
Alleles Animal Model Automobile Driving Binding Biological Assay CRISPR screen Cells Complex Deoxyribonucleases Development Disease Elements Encyclopedia of DNA Elements Enhancers Epithelial Etiology Functional disorder Gene Expression Genes Genetic Transcription Genetic Variation Genome Goals Health Heritability Human Human Genome Hypersensitivity Individual Location Maps Measurement Measures Methods Molecular Mus Mutagenesis Neurons Phenotype Physiological Population Regulatory Element Reporter Screening Result Sequence Homology Sulfur Untranslated RNA Variant Writing base causal variant chromatin modification comparative direct application disorder risk genome wide association study genome-wide human disease human model human pluripotent stem cell improved induced pluripotent stem cell insight molecular phenotype success trait

项目摘要

PROJECT SUMMARY Recent advances to characterize cis-regulatory elements (CRE), including massively parallel reporter assays and CRISPR-based screens of non-coding elements, have transformed our ability to comprehensively characterize the non-coding genome at scale. Large scale efforts by us and others through the Encyclopedia of DNA Elements (ENCODE) consortium are now underway to apply these methods genome-wide across many cellular states. The results of these screens will have a transformative impact on our ability to read and write the regulatory grammar of the cell. One direct application will be in the interpretation of causal alleles for human disease risk and other phenotypic traits identified through genome-wide association studies. From these studies we now know the majority of heritability for complex traits resides in non-coding regions of the genome. Until recently it has been difficult to pinpoint individual causal alleles but progress is now being made to identify and elucidate their molecular function. Despite our burgeoning success in understanding how a variant impacts molecular phenotypes (e.g. gene transcription), we lack the ability to systematically evaluate allele(s) within model organisms to understand their impact on physiological function. This disconnect is partially due to our inability to identify the homologous non-coding region to target within model organisms. To aid in modeling human regulatory variation in the mouse, in this project we will develop improved maps of homologous CREs between human and mouse. Current comparative approaches rely on sequence homology and correlative measures of gene expression such as regions of DNase hypersensitivity and chromatin modifications. While these methods have provided valuable insight, they lack direct quantitative measurements of a CRE's impact on individual genes and the location of the cis-regulatory modules (CRMs) within the CREs responsible for activity. To overcome these shortcomings, in this study we will develop maps of CRE conservation based directly on function. To accomplish this, we will differentiate induced pluripotent stem cells (iPSCs) from human and mouse to early developmental states as the starting material for screens of CRE activity. We will use (i) a CRISPR-based screen to endogenously perturb putative CREs important for neuronal and epithelial function; and (ii) CREs with concordant and discordant activity across the two species will then undergo saturation mutagenesis using a massively parallel reporter assay (MPRA). Results from the MPRA will identify CRMs (e.g. TF binding motifs) within each CRE driving regulatory activity of the element. We will use the results from both screens to construct improved maps of CRE conservation that will inform how to copy the effects of genetic variation residing at these regions across species. Doing so will accelerate our progress in moving human disease variants into animal models, thereby allowing us to better understand the pathophysiology of complex diseases in the human population.

项目概要表征顺式调控元件 (CRE) 的最新进展，包括大规模并行报告基因检测和基于 CRISPR 的非编码元件筛选，改变了我们综合分析的能力大规模表征非编码基因组。我们和其他人通过百科全书进行的大规模努力 DNA Elements (ENCODE) 联盟目前正在将这些方法应用于许多基因组范围内细胞状态。这些屏幕的结果将对我们的阅读和写作能力产生革命性的影响细胞的调节语法。一个直接的应用是解释因果等位基因通过全基因组关联研究确定的人类疾病风险和其他表型特征。从通过这些研究，我们现在知道复杂性状的大部分遗传力存在于非编码区域基因组。直到最近，还很难确定单个因果等位基因，但现在正在取得进展识别和阐明它们的分子功能。尽管我们在理解如何变异影响分子表型（例如基因转录），我们缺乏系统评估的能力模型生物体内的等位基因，以了解它们对生理功能的影响。这种断开连接是部分原因是我们无法识别模型生物体内的同源非编码区域。到帮助模拟小鼠的人类调节变化，在这个项目中，我们将开发改进的图谱人类和小鼠之间的同源 CRE。当前的比较方法依赖于序列同源性以及基因表达的相关测量，例如 DNase 超敏区域和染色质修改。虽然这些方法提供了有价值的见解，但它们缺乏直接的定量测量 CRE 对个体基因的影响以及 CRE 中顺式调控模块 (CRM) 的位置负责活动。为了克服这些缺点，在本研究中我们将开发 CRE 地图直接基于函数的守恒。为了实现这一目标，我们将分化诱导多能干细胞（iPSC）从人类和小鼠到早期发育状态，作为 CRE 筛选的起始材料活动。我们将使用 (i) 基于 CRISPR 的筛选来内源性扰乱对神经元重要的推定 CRE 和上皮功能； (ii) 两个物种之间活动一致和不一致的 CRE 将使用大规模并行报告分析（MPRA）进行饱和诱变。 MPRA 的结果将识别每个 CRE 中驱动元件监管活动的 CRM（例如 TF 结合基序）。我们将使用两个屏幕的结果构建改进的 CRE 保护地图，以告知如何复制这些区域的遗传变异对物种的影响。这样做会加速我们的进步将人类疾病变异转移到动物模型中，从而使我们能够更好地了解人类复杂疾病的病理生理学。