Functionally Assessing Transcriptional Enhancers In Vivo

项目总结 数十万远程作用的增强子通过编排控制人类基因组的功能 基因转录在出生前和出生后的发育以及细胞和疾病状态下的转录 纸巾。它们表现出显著的细胞类型特异性和动态的空间和时间活动模式。而当 现在有大量的间接证据表明，增强子的序列变化可能会对 关于人类表型变异和许多疾病过程，我们对生物功能的理解 在增强子序列中编码的序列仍然不完整。这是一个重大挑战， 全基因组测序(WGS)观察到的患者和患者增强子序列变异的解释 用于将增强子内的序列变异与疾病和其他表型联系起来。在过去16年里， 此R01支持的研究计划提供了对增强剂生物学和开创性的重大见解 用于增强子发现和表征的工具。这包括第一次演示来自 用于增强子发现的哺乳动物组织，以及一套不断进化的小鼠工程方法 在体内研究增强剂。这个项目还产生了最大的人类和老鼠的凝聚力集合 到目前为止可用的活体特征增强器，并将Vista增强程序浏览器作为主要 社区资源。在此研究计划的下一阶段，我们建议利用独特的功能 以前在该计划下开发的，以进一步促进我们对一般增强子生物学的理解，以 补充最近建立的NHGRI基因组变异对功能的影响(IGVF)的工作 与急需的增强子变体体内评估的联盟，并提供增强剂资源 为更广泛的社区服务。我们的具体目标包括：(1)探索增强子的内部解剖 通过大规模突变与体内小鼠试验相结合的序列。我们还将删除大型非 从小鼠基因组编码基因组间隔(基因沙漠)来确定广泛的功能意义 超越单个增强子元件的非编码DNA。(2)作为IGVF联盟的附属机构工作， 我们将对人类变异进行大规模的体内探索，并预测对增强子功能的影响 通过我们独特的鼠标工程能力评估人类的变异/突变。这将包括小心 大规模平行报告分析(MPRA)和CRISPR筛查的验证和校准 与活体小鼠的报告分析相比较。(3)我们将继续提供体内小鼠检测 并通过Vista增强浏览器提供我们的结果。总而言之，在 在该计划的下一阶段，我们希望提供对促进剂生物学的有影响力的见解，作为主要的 非编码基因组功能的类别，应用创新的工具来展示变体在体内的影响 由IGVF联盟确定，并继续提供独特的增强子分析工具和资源 对更广泛的社区而言。

PROJECT SUMMARY Hundreds of thousands of distant-acting enhancers control the function of the human genome by orchestrating the transcription of genes during pre- and postnatal development and in normal and disease states of cells and tissues. They display remarkable cell type specificity and dynamic spatial and temporal activity patterns. While there is now abundant indirect evidence that sequence changes in enhancers are likely to impact substantially on human phenotypic variation and many disease processes, our understanding of how biological function is encoded within enhancer sequences remains incomplete. This represents a major challenge for the interpretation of variation in enhancer sequences observed by whole-genome sequencing (WGS) in patients and for linking sequence variants within enhancers to diseases and other phenotypes. Over the past 16 years, the research program supported by this R01 has provided major insights into enhancer biology and groundbreaking tools for enhancer discovery and characterization. This included the first demonstration of ChIP-seq from mammalian tissues for enhancer discovery, and a constantly evolving suite of mouse engineering methods for studying enhancers in vivo. This program has also produced the largest cohesive collection of human and mouse in vivo-characterized enhancers available to date and provides the VISTA Enhancer Browser as a major community resource. In the next phase of this research program, we propose to leverage the unique capabilities previously developed under this program to further advance our understanding of general enhancer biology, to complement the work of the recently established NHGRI Impact of Genomic Variation on Function (IGVF) consortium with critically needed in vivo assessments of enhancer variants, and to provide enhancer resources for the extended community. Our specific aims include: (1) We will explore the inner anatomy of enhancer sequences through large-scale mutagenesis coupled to in vivo mouse assays. We will also delete large non- coding genome intervals (gene deserts) from the mouse genome to determine the broad functional significance of non-coding DNA beyond individual enhancer elements. (2) Working as Affiliates with the IGVF Consortium, we will perform large-scale in vivo exploration of human variants with predicted impact on enhancer function to assess human variation/mutation through our unique mouse engineering capabilities. This will include careful validation and calibration of massively parallel reporter assays (MPRAs) and CRISPR screens through comparison with in vivo mouse reporter assays. (3) We will continue to provide access to in vivo mouse assays for the community and make our results available through the VISTA Enhancer Browser. In combination, in the next phase of this program we expect to deliver impactful insights into the biology of enhancers as a major category of non-coding genome function, apply innovative tools to demonstrate the in vivo impact of variants identified by the IGVF consortium, and continue to make unique enhancer analysis tools and resources available to the broader community.