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

• 批准号: 10491357
• 负责人: Ryan Tewhey
• 金额: $ 51.92万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 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/10491357
• 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; 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

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.

项目摘要 表征顺式调节元件（CRE）的最新进展，包括大量平行的报告基因测定 以及基于CRISPR的非编码元素的屏幕，已经改变了我们全面的能力 在大规模表征非编码基因组。我们和他人通过百科全书的大规模努力 DNA元素（编码）联盟正在进行中，以在许多基因组中应用这些方法 细胞状态。这些屏幕的结果将对我们读写能力产生变革性的影响 细胞的调节语法。一个直接应用将是解释因果等位基因的 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 这些研究现在知道，复杂性状的大部分遗传力都存在于非编码区域 基因组。直到最近，很难确定单个因果等位基因，但现在正在取得进展 识别和阐明其分子功能。尽管我们在了解如何 变体影响分子表型（例如基因转录），我们缺乏系统评估的能力 模型生物中的等位基因了解其对生理功能的影响。此断开连接是 部分原因是我们无法确定同源的非编码区域以靶向模型生物体。到 有助于对小鼠的人体调节变化进行建模，在此项目中，我们将开发改进的地图 人与小鼠之间的同源板。当前的比较方法取决于序列同源性 以及基因表达的相关度量，例如DNase超敏反应和染色质区域 修改。尽管这些方法提供了宝贵的见解，但它们缺乏直接的定量测量 CRE对单个基因的影响和CIS调节模块（CRMS）的位置 负责活动。为了克服这些缺点，在这项研究中，我们将开发CRE的地图 直接基于功能保护。为此，我们将区分诱导的多能干细胞 （IPSC）从人和小鼠到早期发育状态，作为CRE屏幕的起始材料 活动。我们将使用（i）基于CRISPR的屏幕对神经元很重要的内源性扰动推定CRE 和上皮功能； （ii）在这两个物种中具有一致和不一致的活动的CRE将会 使用大量平行的报告基因测定法（MPRA）进行饱和诱变。 MPRA的结果 将在每个CRE驱动该元件的调节活动中识别CRM（例如TF结合基序）。我们将 使用两个屏幕的结果来构建改进的CRE保护地图，这将告知如何复制 跨物种的这些区域存在遗传变异的影响。这样做会加速我们的进步 在将人类疾病变体转移到动物模型中，从而使我们能够更好地了解 人口中复杂疾病的病理生理学。