Spatiotemporal mapping of enhancer activity in developing frog embryos

青蛙胚胎发育中增强子活性的时空图谱

基本信息

批准号：
10511083
负责人：
Ken W.Y. Cho
金额：
$ 22.82万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-19 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10511083
关键词：
Address Adult Algorithms Amphibia Animals Behavior Binding Sites Biological Biological Assay Biological Models Biological Process Cell Lineage Cells Characteristics Collection Complex Computational Biology Data Data Analyses Development Distal Embryo Embryonic Development Enhancers Event Gastrula Gene Expression Gene Expression Profile Genes Genetic Diseases Genetic Transcription Genomics Goals Health Heterogeneity Human Individual Internet Mammals Maps Measurement Methods Molecular Mutate Mutation Nucleic Acid Regulatory Sequences Organism Pathway interactions Pattern Phylogenetic Analysis Plant Roots Population Positioning Attribute Process Rana Regulation Regulator Genes Resolution Role Signal Transduction Specific qualifier value Structure Sum Surveys System Tetrapoda Tissue-Specific Gene Expression Tissues Transcriptional Regulation Vertebrates Work Xenopus blastomere structure cell type experimental study gastrulation genetic information genome-wide innovation insight mammalian genome programs reconstruction single-cell RNA sequencing spatiotemporal stem success transcription factor transcriptomics vertebrate genome zygote

项目摘要

Project Summary: All cell types in an organism share the same basic genetic information, yet they execute remarkably diverse gene expression programs and behaviors. Much of this diversity is derived from cell type-specific and condition-specific usage of gene-distal regulatory regions known as cis-regulatory modules (CRMs) (also known as enhancers). Typical mammalian genomes contain hundreds of thousands of CRMs distributed across large genomic distances that act collectively and collaboratively to produce differential gene expression patterns of extreme complexity. At present, our understanding of the biological roles of most of these CRMs remains limited. For instance, how are specific CRMs selected to regulate a given gene's expression? How do different CRMs coordinate and activate various target genes in a spatially and temporally regulated manner? Our lack of understanding is in large part due to current limitations in delivering simultaneous experimental measurements of the activities of large numbers of CRMs during animal development. The major goal of this proposal is the successful use of Xenopus gastrula-stage embryos as a model system for quantifying CRM activities involved in delivering precise spatiotemporal expression patterns of target genes. While CRM activities are to be assayed at a genome-wide level, their associated gene regulatory mechanisms would be determined with single-cell resolution. Applying advanced computational biology algorithms to that data will deliver mapping of CRM activities sufficient to infer the TFs and associated signaling processes involved in regulation of differential cell states in gastrulae at single-cell resolution. We propose to address two specific aims. First, we will apply a modified STARR-seq approach to identify CRMs that regulate spatiotemporal gene expression patterns in gastrula embryos. We plan to mutate potential TF binding sites within CRMs to aid in identifying their biological functions. Second, we will combine scRNA-seq with STARR-seq as a means of uncovering CRM-centric gene regulatory structures critical for specifying cell states for every cell in gastrula-stage embryos. In sum, we will use Xenopus tropicalis gastrula-stage embryos as the model system for generating a system-level understanding of CRM activities in single cells because of the key phylogenetic position amphibians occupy in the vertebrate evolutionary lineage. The approach proposed here would be much more difficult to perform in mammals, because their embryos are not easily accessed for certain necessary experimental manipulations, and because mammalian embryo availability is rate limiting for some genomic work.

项目概要：生物体中的所有细胞类型都共享相同的基本遗传信息，但它们的执行能力却非常出色不同的基因表达程序和行为。这种多样性很大程度上源自细胞类型特异性以及基因远端调节区域（称为顺式调节模块（CRM））的特定条件使用（也称为增强剂）。典型的哺乳动物基因组包含数十万个 CRM 分布在较大的基因组距离上，集体和协作地产生差异极其复杂的基因表达模式。目前，我们对生物作用的认识大多数 CRM 仍然有限。例如，如何选择特定的 CRM 来监管给定的基因的表达？不同的CRM如何在空间上协调和激活各种靶基因和临时调节方式？我们缺乏了解很大程度上是由于目前的局限性期间提供大量 CRM 活动的同步实验测量动物发育。该提案的主要目标是成功利用非洲爪蟾原肠阶段胚胎作为模型系统，用于量化涉及提供精确时空的 CRM 活动目标基因的表达模式。虽然 CRM 活动要在全基因组水平上进行分析，它们相关的基因调控机制将通过单细胞分辨率来确定。正在申请该数据的先进计算生物学算法将提供足够的 CRM 活动映射推断参与调节差异细胞状态的 TF 和相关信号传导过程单细胞分辨率的原肠胚。我们建议解决两个具体目标。首先，我们将应用修改后的 STARR-seq 方法识别调节原肠胚时空基因表达模式的 CRM 胚胎。我们计划突变 CRM 中潜在的 TF 结合位点，以帮助识别其生物学特性功能。其次，我们将 scRNA-seq 与 STARR-seq 结合起来，作为发现以 CRM 为中心的方法基因调控结构对于指定原肠胚阶段胚胎中每个细胞的细胞状态至关重要。在总之，我们将使用热带爪蟾原肠胚阶段胚胎作为模型系统来生成由于关键的系统发育位置，对单细胞中 CRM 活动的系统级理解两栖动物占据脊椎动物的进化谱系。这里提出的方法将是很多在哺乳动物身上进行起来更加困难，因为它们的胚胎在某些情况下不容易获得必要的实验操作，并且因为哺乳动物胚胎的可用性受到限制一些基因组工作。