Deciphering real-time dynamics of the human genome organization in response to DNA damage and gene expression

项目总结 人类基因组被高度组织和调控，以表达细胞类型和组织特有的基因。在.期间 间期，染色体在细胞核中占据不同的区域，称为染色体区域，这是一个概念 早在1885年就提出了，并于1982年进行了演示。过去二十年的技术发展 已经允许对基因组三维结构的变化进行检查和建模。但 了解在细胞核中定位或移动染色体位点的机制将需要高度的- 实时研究分辨率。CRISPRain弓的最新发展允许同时标记， 在活的人类细胞中，以高分辨率可视化和实时跟踪多达七个特定的基因组位点。 初步研究表明，同源和非同源染色体上的基因座移动的方式不同 速度、方向和限制。CRISPRainrow允许对运动进行量化分类 不同的基因座，检测DNA双链断裂时的加速移动，以及染色体 整体组织。这项提议的目标是回答以下问题。的空间范围是多少 基因组的基因座运动？是特定于染色体和/或的动态空间范围的维度 取决于它的核内定位吗？染色质区域的远距离迁移是如何发生的 在DNA双链断裂之后？转录和染色质重构体的相互作用是什么 基因组位置的移动和整个染色质组织的变化？在K99阶段，以CRISPR为基础 单分子实时显微镜将被用来定量成像和表征基因组基因座 并检验基因组位置的移动取决于染色体的同一性或 核地点。在单细胞DNA损伤修复过程中，染色质区域的重新定位也将被追踪。 在R00阶段，染色体构象捕获实验将用于分子 描述染色体相互作用对DNA损伤以及分子、细胞和遗传的影响 实验将用于研究染色质对转录激活/沉默的反应动力学， 核小体解体和肿瘤抑制基因P53。这项建议是高度跨学科的，将摆脱 人类基因组的组织和稳定性，并将导致强大的定量实时方法学 目的：探讨癌细胞染色体易位的机制。这项研究借鉴了以下专家的经验 是单分子实时显微镜、核生物学和 基因组/核结构以及谁将帮助屠呦呦为向独立职业生涯的过渡做好准备 调查员。

PROJECT SUMMARY The human genome is highly organized and regulated to express cell-type and tissue-specific genes. During interphase, chromosomes occupy distinct regions in the nucleus, known as chromosome territories, a concept proposed as early as 1885 and demonstrated in 1982. Technological developments over the last two decades have allowed changes in the three-dimensional architecture of the genome to be examined and modeled. But understanding the mechanisms that localize or mobilize chromosomal loci in the nucleus will require high- resolution studies in real time. The recent development of CRISPRainbow allows the simultaneous labeling, visualization, and real-time tracking of up to seven specific genomic loci at high resolution in live human cells. Preliminary studies show that loci on homologous and non-homologous chromosomes move with different speeds, directions, and confinement. CRISPRainbow allows quantitative categorization of the movements of various loci, detects accelerated movements at DNA double-strand breaks, and change in a chromosome's overall organization. The goal of this proposal is to answer the following questions. What is spatial range of genomic loci movements? Is the dimension of the dynamic spatial range chromosome-specific and/or dependent of its intranuclear localization? How does long-range chromatin territory relocation take place following DNA double-strand breaks? What is the interplay of transcription and the chromatin remodelers on genomic loci movements and entire chromatin organization changes? During the K99 phase, CRISPR-based single-molecule real-time microscopy will be used to quantitatively image and characterize genomic loci movements and to test the hypothesis that genomic loci movements depend on chromosome identity or on nuclear location. Chromatin territory relocation will also be tracked during DNA damage repair in single cells. During the R00 phase, chromosome conformation capture experiments will be used to molecularly characterize changes in chromosomal interactions upon DNA damage and molecular, cellular, and genetic experiments will be used to investigate chromatin dynamics in response to transcription activation/silencing, nucleosome disassembly, and the tumor suppressor p53. This proposal is highly interdisciplinary, will shed the light on human genome organization and stability, and will lead to powerful quantitative real-time methodology to investigate mechanisms of chromosome translocation in cancer cells. This study draws on expertise from mentors who are leaders in the field of single-molecule real-time microscopy, nuclear biology, and genome/nuclear architecture and who will help prepare Dr. Tu for a transition to a career as an independent investigator.