Elucidating how pioneer factors drive dynamic chromatin accessibility during zygotic genome activation

阐明先锋因素如何在合子基因组激活过程中驱动动态染色质可及性

• 批准号: 10300009
• 负责人: Mark Pownall
• 金额: $ 4.34万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-01 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10300009
• 关键词: 3-Dimensional; ATAC-seq; Affect; Architecture; Binding; Biological Assay; Cells; Chromatin; Chromatin Structure; Complex; DNA; DNA Methylation; Data; Detection; Development; Disease; Disease Progression; Embryo; Embryonic Development; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Guide RNA; Health; Hi-C; High-Throughput Nucleotide Sequencing; Human; In Situ; Individual; Link; Malignant Neoplasms; Measures; Mediating; Methods; Organism; Outcome; Regulation; Reporter; Resolution; Role; Sampling; Structure; Testing; Time; Totipotent; Visualization; Work; XCL1 gene; Zebrafish; base; chromatin remodeling; egg; falls; histone modification; human disease; in vivo; insight; live cell imaging; mutant; novel; pluripotency; spatiotemporal; sperm cell; temporal measurement; transcription factor; zygote

PROJECT SUMMARY Dynamic remodeling of the three-dimensional organization of the genome regulates gene expression to coordinate the healthy development of organisms and can impact disease progression when dysregulated. Recent advances have enabled the visualization of accessible chromatin in situ in fixed samples; however, we still do not fully understand how changes in chromatin organization occur in vivo during vertebrate development, especially at the single-cell level. Therefore, the goal of this project is to study the dynamic remodeling of chromatin organization during zygotic genome activation, when the transcriptionally silent embryo first initiates transcription of its own genes and genome architecture is dramatically remodeled. First (Aim 1), I will investigate the dynamic spatial organization of chromatin accessibility during genome activation in zebrafish. To do so, I will use Live-ATAC, a method I recently developed. Live-ATAC enables real time detection of chromatin accessibility in vivo at the single-cell level in living embryos. Using this method, I will determine precisely when and where chromatin accessibility is gained as the embryo undergoes genome activation and transcription is initiated. Second (Aim 2), I will use Live-ATAC in mutant embryos to dissect the role of pioneer transcription factors in dynamically remodeling chromatin accessibility during genome activation. Specifically, I will use embryos lacking functional Nanog, Oct4, and Sox19b which are maternally supplied transcription factors have been implicated in pluripotency and genome activation. Simultaneously, I will measure transcription of the first zygotically transcribed genes to determine the causal relationships between transcription factors, chromatin accessibility, and transcription in real time in vivo. Altogether, this work will provide an unprecedented view of how chromatin organization is dynamically remodeled during cellular reprogramming and begin elucidating the underlying mechanisms. This will inform our understanding of how three-dimensional genome organization may affect human embryogenesis and disease given the likely conserved regulatory principles.

项目总结 基因组三维组织的动态重塑调节基因表达以 协调生物体的健康发展，并在失调时影响疾病的进展。 最近的进展使得在固定样本中原位显示可接近的染色质成为可能；然而，我们 仍然不完全了解在脊椎动物体内染色质组织的变化是如何发生的 发展，特别是在单细胞水平上。因此，本项目的目标是研究这一动态 合子基因组激活期间染色质组织的重塑，当转录沉默时 胚胎首先启动自身基因的转录，基因组结构被戏剧性地重塑。第一 (目标1)，我将研究基因组激活过程中染色质可及性的动态空间组织 在斑马鱼身上。为此，我将使用Live-ATAC，这是我最近开发的一种方法。实时-ATAC可实现实时 活体胚胎单细胞水平染色质可及性检测。使用这种方法，我将 准确地确定胚胎在何时何地获得了染色质的可及性 激活和转录被启动。第二个(目标2)，我将在突变胚胎中使用Live-ATAC来解剖 先锋转录因子在基因组动态重塑染色质可及性中的作用 激活。具体地说，我将使用缺乏功能的Nanog、Oct4和Sox19b的胚胎，这些胚胎是母体的 提供的转录因子与多能性和基因组激活有关。同时，我 将测量第一个合子转录的基因的转录，以确定因果关系 转录因子之间，染色质的可及性，以及体内的实时转录。总而言之，这 这项工作将提供一个前所未有的视角，了解染色质组织是如何在 细胞重新编程，并开始阐明潜在的机制。这将使我们了解到 三维基因组组织如何影响人类胚胎发育和疾病 保守的监管原则。