Unraveling the Telomere Black Box: A New Single-Molecule Approach to Define the Telomere Chromatin Landscape and its Functional Mechanisms

揭开端粒黑匣子：定义端粒染色质景观及其功能机制的新单分子方法

• 批准号: 10471552
• 负责人: Ci Ji Lim
• 金额: $ 138.56万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10471552
• 关键词: Area; Binding; Biological Process; Biology; Black race; Cell Cycle; ChIP-seq; Chromatin; Chromosomes; DNA; DNA Methylation; DNA Structure; DNA biosynthesis; DNA sequencing; DNA-Protein Interaction; Genomic DNA; Genomic Instability; Genomic Segment; Human; Human Chromosomes; Imaging technology; Knowledge; Left; Length; Malignant Neoplasms; Maps; Methodology; Nature; Outcome; Output; Premature aging syndrome; Process; Proteins; Research; Rest; Techniques; Telomere Capping; Telomere Length Maintenance; Variant; experimental study; genome integrity; human disease; innovation; interest; non-Native; novel; prevent; single molecule; telomere; telomere loss; tool

Summary Telomeres are end-capping protein-DNA structures at the ends of the linear human chromosomes. They protect our genome integrity by sacrificing their repetitive DNA when the ends of the chromosome suffer attrition during DNA replication and camouflaging the chromosome ends from wrong DNA breakage recognition. Deregulation or loss of telomeres results in genome instability and leads to human diseases such as cancer and premature aging. The telomere's repetitive DNA nature provides a unique challenge in understanding their biological processes. This is because telomeric proteins can bind the repetitive telomeric DNA in many ways, leading to complexity and diversity in the telomere chromatin landscape; there are functional consequences to how telomeric proteins decorate a telomere chromatin landscape because these proteins directly participate in telomere protection and length maintenance. Thus, our understanding of telomeres is like a "black box". We know the inputs (proteins and lncRNA) and outputs (telomere length and end-protection) and understand how variations of inputs transform to output changes. However, we do not know what is going on inside the "black box". This "black box" is the telomere chromatin landscape. Characterizing the telomere chromatin landscape has been an insurmountable task for the telomere research field for decades. The ChIP-Seq technique has revolutionized chromosome biology research, but repetitive genomic regions such as the telomeres are left behind. This is because the relative positional information of the protein-DNA interactions is lost upon the fragmentation step in ChIP-Seq, preventing us from reconstructing the chromatin landscape of interest. This proposal seeks to innovate new tools to map the human telomere chromatin landscape at a single-telomere level. These tools will then use to study how the telomere chromatin landscape regulates telomere end-protection and length maintenance. First, I will establish the proof-of-concept experiments for using non-native DNA methylation to mark protein-DNA interactions at the repetitive telomeric DNA regions and reconstruct the chromatin landscapes with structural details. These tools will then be used to tackle two major research areas: (1) What is the human telomere chromatin landscape and how it changes across the cell cycle from a resting protective state to one permissive to DNA replication progression. (2) How changes in the human telomere chromatin landscape drive telomere length maintenance. This proposal thus consists of both technological and conceptual innovations. The new tools will provide a new way to investigate chromosome biology at repetitive genomic DNA regions; thus, its impact extends beyond the telomeres. We will get an unprecedented first look into the telomere chromatin landscape. Hence, this proposal has enormous potential to open multiple new research directions in telomere biology; a paradigm shift in our telomere knowledge is expected. Because of the biomedical importance of telomeres, the outcome of this proposal can provide novel avenues to tackle telomere-related human diseases.

总结 端粒是位于线性人类染色体末端的封端蛋白质-DNA结构。他们保护 我们的基因组的完整性，牺牲他们的重复DNA时，染色体的两端遭受磨损， DNA复制和染色体末端的断裂识别错误。放松管制 端粒的缺失导致基因组不稳定，并导致人类疾病，如癌症和早产儿。 衰老端粒的重复DNA性质为理解它们的生物学特性提供了独特的挑战。 流程.这是因为端粒蛋白可以以多种方式结合重复的端粒DNA，从而导致 端粒染色质景观的复杂性和多样性;有功能的后果，如何 端粒蛋白质装饰端粒染色质景观，因为这些蛋白质直接参与 端粒保护和长度维持。因此，我们对端粒的理解就像一个“黑匣子”。我们 知道输入（蛋白质和lncRNA）和输出（端粒长度和末端保护），并了解如何 输入的变化转化为输出的变化。然而，我们不知道“黑”里面是怎么回事 盒子”。这个“黑盒子”就是端粒染色质景观。 端粒染色质景观的表征一直是端粒研究中难以克服的难题 几十年来的田野ChIP-Seq技术彻底改变了染色体生物学研究，但重复性 基因组区域如端粒被留下。这是因为， 在ChIP-Seq中的片段化步骤中，蛋白质与DNA的相互作用消失，使我们无法重建蛋白质与DNA的相互作用。 染色质景观。该提案旨在创新新的工具来绘制人类端粒染色质 在一个端粒水平的景观。然后，这些工具将用于研究端粒染色质景观 调节端粒末端保护和长度维持。首先，我将建立概念验证 使用非天然DNA甲基化标记重复端粒处的蛋白质-DNA相互作用的实验 DNA区域和重建染色质景观与结构细节。这些工具将用于 解决两个主要的研究领域：（1）人类端粒染色质景观是什么以及它如何变化 在细胞周期中从静止保护状态到允许DNA复制进程的状态。(2)如何 人类端粒染色质景观的变化驱动端粒长度的维持。 因此，这一建议既包括技术创新，也包括概念创新。新工具将提供 研究重复基因组DNA区域的染色体生物学的新方法;因此，其影响超越了 端粒我们将得到一个前所未有的第一次看端粒染色质景观。所以这 该提案具有巨大的潜力，可以在端粒生物学中开辟多个新的研究方向;一个范式转变 我们的端粒知识是预期的。由于端粒在生物医学上的重要性， 该提案可以为解决端粒相关的人类疾病提供新的途径。