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

项目摘要 表征顺式调控元件（CRE）的最新进展，包括大规模平行报告分析 和基于CRISPR的非编码元件筛选，已经改变了我们全面 大规模地描述非编码基因组。我们和其他人通过百科全书的大规模努力 DNA元件（ENCODE）联盟目前正在进行中，以将这些方法应用于许多基因组范围 细胞状态这些屏幕的结果将对我们的读写能力产生变革性的影响 细胞的调节语法。一个直接的应用将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性状。从 通过这些研究，我们现在知道，复杂性状的大部分遗传力位于基因组的非编码区。 基因组直到最近，还很难确定个别的致病等位基因，但现在正在取得进展 以鉴定和阐明它们的分子功能。尽管我们在理解一个 变异影响分子表型（例如基因转录），我们缺乏系统评估 等位基因在模式生物体中，以了解其对生理功能的影响。与这种脱节 部分原因是我们不能鉴定出模式生物体内靶向的同源非编码区。到 帮助在小鼠中模拟人类调节变异，在这个项目中，我们将开发改进的地图， 人和小鼠之间同源克雷斯。目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DNA酶超敏区域和染色质 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对单个基因的影响以及克雷斯内顺式调节模块（CRM）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地图 直接基于功能的保护。为了实现这一点，我们将分化诱导多能干细胞 作为筛选CRE的起始材料， 活动我们将使用（i）基于CRISPR的筛选来内源性干扰对神经元细胞增殖重要的推定的克雷斯。 和上皮功能;以及（ii）在两个物种中具有一致和不一致活性的克雷斯将 使用大规模平行报告基因测定（MPRA）进行饱和诱变。MPRA的结果 将鉴定每个CRE内驱动元件调节活性的CRM（例如TF结合基序）。我们将 使用两个屏幕的结果来构建CRE保护的改进地图，该地图将告知如何复制 存在于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。

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.