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

摘要 端粒是人类线性染色体末端的蛋白质-DNA结构。他们保护 我们的基因组完整性，通过牺牲他们的重复DNA时，染色体末端遭受磨损 DNA复制和伪装来自错误的DNA断裂识别的染色体末端。放松管制 或端粒丢失会导致基因组不稳定，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，导致 端粒染色质景观中的复杂性和多样性；端粒染色质如何 端粒蛋白质装饰着端粒染色质的景观，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 了解输入(蛋白质和lncRNA)和输出(端粒长度和末端保护)，并了解如何 投入的变化转化为产出的变化。然而，我们不知道“黑色”里面发生了什么 这个“黑匣子”就是端粒染色质景观。 端粒染色质图谱的表征一直是端粒研究中一项不可克服的任务 几十年来一直在田野里。芯片序列技术使染色体生物学研究发生了革命性的变化，但具有重复性 像端粒这样的基因组区域被留在了后面。这是因为 蛋白质-DNA相互作用在CHIP-SEQ的碎裂步骤中丢失，阻止我们重建 感兴趣的染色质景观。这项提议旨在创新绘制人类端粒染色质图谱的新工具 在单个端粒水平上的景观。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 利用非天然DNA甲基化标记重复端粒上蛋白质-DNA相互作用的实验 DNA区域，并用结构细节重建染色质景观。然后，这些工具将用于 解决两个主要研究领域：(1)什么是人类端粒染色质景观及其变化 在细胞周期中，从静止的保护状态到允许DNA复制的状态。(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，这项提议既包括技术创新，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，它的影响超出了 端粒。我们将史无前例地第一次看到端粒染色质的景观。因此，这是 提议有巨大的潜力在端粒生物学中开辟多个新的研究方向；范式的转变 在我们的端粒中，知识是可以期待的。由于端粒在生物医学上的重要性，这一结果 该提议可以为解决端粒相关的人类疾病提供新的途径。

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.