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

解读人类基因组组织响应 DNA 损伤和基因表达的实时动态

• 批准号: 9432293
• 负责人: Li-Chun Tu
• 金额: $ 9万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9432293
• 关键词: Affect; Architecture; Bacteria; Behavior; Binding Sites; Biology; CRISPR/Cas technology; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Chromosomal translocation; Chromosome Territory; Chromosome abnormality; Chromosomes; Chromosomes, Human, 16-18; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA-Binding Proteins; Data; Development; Development Plans; Dimensions; Disease; Distant; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genome Stability; Genomics; Goals; Histone Deacetylase; Human; Human Genome; Image; Imagery; Individual; Interphase; Interphase Chromosome; Label; Lead; Light; Location; MDM2 gene; Malignant Neoplasms; Measures; Mentors; Methodology; Methods; Microscopy; Modeling; Molecular; Molecular Conformation; Motion; Movement; Mutation; Normal Cell; Nuclear; Nuclear Lamin; Nucleoplasm; Nucleosomes; Organizational Change; Paint; Pattern; Phase; Process; Property; Protein p53; Regulatory Element; Research; Research Personnel; Resolution; Speed; TP53 gene; Techniques; Testing; Time; Tissues; Transcriptional Activation; Transcriptional Regulation; Transgenes; base; cancer cell; career; career development; cell type; chromosome conformation capture; experimental study; improved; knock-down; novel strategies; nuclease; overexpression; physical model; quantitative imaging; response; single molecule; transcription factor; whole genome

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.

项目概要 人类基因组经过高度组织和调节，可表达细胞类型和组织特异性基因。期间 间期，染色体占据细胞核中不同的区域，称为染色体区域，这是一个概念 早在 1885 年就被提出，并于 1982 年得到论证。过去二十年的技术发展 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动员染色体位点的机制将需要高度的 实时分辨率研究。 CRISPRainbow 最近的发展允许同时标记， 可视化，并以高分辨率实时跟踪活人类细胞中多达七个特定基因组位点。 初步研究表明，同源和非同源染色体上的位点移动方式不同。 速度、方向和限制。 CRISPRainbow 可以对运动进行定量分类 各种位点，检测 DNA 双链断裂处的加速运动以及染色体的变化 整体组织。该提案的目标是回答以下问题。什么是空间范围 基因组位点移动？动态空间范围的维度是染色体特异性的和/或 取决于其核内定位？长距离染色质区域迁移是如何发生的 DNA双链断裂后？转录和染色质重塑之间的相互作用是什么？ 基因组位点运动和整个染色质组织发生变化？在 K99 阶段，基于 CRISPR 的 单分子实时显微镜将用于定量成像和表征基因组位点 运动并检验基因组位点运动取决于染色体身份或 核位置。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生物学 描述 DNA 损伤和分子、细胞和遗传导致的染色体相互作用变化的特征 实验将用于研究响应转录激活/沉默的染色质动力学， 核小体分解和肿瘤抑制因子 p53。该提案具有高度跨学科性，将摆脱 阐明人类基因组的组织和稳定性，并将带来强大的定量实时方法 研究癌细胞染色体易位的机制。这项研究借鉴了来自以下机构的专业知识 导师是单分子实时显微镜、核生物学和 基因组/核架构以及谁将帮助屠博士做好向独立职业生涯过渡的准备 研究者。