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

项目摘要 基因组三维结构的动态重塑调节基因表达， 协调生物体的健康发育，当调节失调时可能会影响疾病的进展。 最近的进展使固定样品中可接近的染色质原位可视化成为可能;然而， 仍然不能完全理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。因此，本项目的目标是研究 在合子基因组激活过程中，当转录沉默时， 胚胎首先启动其自身基因的转录，基因组结构被显著重塑。第一 (Aim 1），我将研究在基因组激活过程中染色质可及性的动态空间组织 在斑马鱼中。为此，我将使用我最近开发的Live-ATAC方法。Live-ATAC支持真实的时间 在活胚胎中在单细胞水平上检测体内染色质可及性。通过这种方法，我将 精确地确定当胚胎经历基因组改造时， 启动激活和转录。第二（目标2），我将在突变胚胎中使用Live-ATAC来解剖 先驱转录因子在基因组过程中动态重塑染色质可及性中的作用 activation.具体来说，我将使用缺乏功能性Nanog、Oct 4和Sox 19 b的胚胎，这些胚胎是母系的。 所提供的转录因子与多能性和基因组活化有关。同时，我 将测量第一个合子转录基因的转录，以确定因果关系 转录因子、染色质可及性和体内真实的转录之间的关系。总之，这 这项工作将提供一个前所未有的观点，染色质组织是如何动态重塑过程中， 细胞重编程并开始阐明潜在的机制。这将使我们了解， 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。

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.