The role of telomeres in 3D organization of the genome within the nucleus

拟议研究项目的目标是了解端粒在细胞核内的定位如何调节正常细胞以及疾病状态和细胞衰老过程中的染色体结构和基因组组织。人类基因组被组织成细胞核内的染色体区域，其中染色体占据确定的体积以促进诸如转录、基因表达、DNA修复和重组的过程。尽管在过去的25年里对细胞核的结构组织进行了深入的研究，但仍然完全不清楚染色体区域如何在细胞分裂和细胞寿命中建立和维持。具体来说，很少有人知道这些领土是如何重建后，染色体解凝聚后有丝分裂。端粒，核蛋白复合物，构成真核生物线性染色体的物理末端，用于掩盖暴露的DNA被识别为双链断裂。人类端粒已被证明定位于细胞核周边，特别是在有丝分裂后，当细胞核重新组装。因此，端粒锚定到包膜上可能是重组染色质结构域的一种可能方式。我将采用Hi-C，染色体构象捕获（3C）的延伸，随后进行高通量测序，以确定端粒和基因组区域之间的相互作用，在一个公正的，全基因组的方式。这将使我能够研究核组织和长距离染色质相互作用如何在细胞周期中发生变化，并在细胞分裂后重新建立。此外，还将研究细胞衰老过程中和具有疾病突变的细胞中的变化。最后，我将解决物理端粒拴系的分子机制，以便能够直接测试端粒锚定在决定基因组结构和组织中的作用。重要的是，区域的变化将代表在衰老、细胞转化或疾病状态期间改变全基因组转录特征和整个细胞核的有效方式。因此，预计拟议的工作将有助于更详细地了解这些进程。我认为，年龄相关疾病的协同发病可能是由于未能有效建立领土，从而改变了整个细胞核的转录途径。我们的目标是确定建立和维持染色体区域所需的分子途径，最终目标是抵消这些途径的病理变化，从而预防疾病的发生。

The goal of the proposed research project is to understand how telomere localization within the nucleus regulates chromosome structure and genome organization, both in normal cells as well as in disease states and during the process of cellular aging. The human genome is organized into chromosome territories within the nucleus, in which chromosomes occupy a defined volume to facilitate processes such as transcription, gene expression, DNA repair and recombination. Despite intense research into the structural organization of the nucleus during the past 25 years, it is still entirely unclear how chromosome territories are established and maintained throughout cell divisions and cellular lifespan. Specifically, very little is known about how these territories are re-established following chromosome decondensation post mitosis. Telomeres, nucleoprotein complexes that make up the physical ends of eukaryotic linear chromosomes, serve to mask the exposed DNA from being recognized as double-strand breaks. Human telomeres have been shown to localize to the nuclear periphery specifically after mitosis, when the nucleus reassembles. Telomere anchoring to the envelope could therefore present one possible way to reorganize chromatin domains. I will employ Hi-C, an extension of chromosome conformation capture (3C) that is followed by high-throughput sequencing, to determine interactions between telomeres and genomic regions in an unbiased, genome-wide manner. This will allow me to investigate how nuclear organization and long-range chromatin interactions are changed during the cell cycle and reestablished after cell division. In addition, changes during cellular aging and in cells with disease mutations will be investigated. Last, I will address the molecular mechanisms of the physical telomere tethering in order to be able to directly test the role of telomere anchoring in the determination of genome architecture and organization. Importantly, changes in territories would represent an efficient way for altering genome wide transcription signatures and the whole nucleus during aging, cellular transformation, or in disease states. The proposed work is therefore expected to contribute to a more detailed understanding of these processes. I propose that it is possible that the synergistic onset of age-associated diseases could be a result of failing to establish territories efficiently, thereby changing transcription pathways throughout the nucleus. Our goal is to identify the molecular pathways that are required for the establishment and maintenance of chromosomal territories, with the ultimate goal of counteracting pathologic changes in these pathways, and thereby preventing the onset of disease.