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

这项拟议的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、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，其中染色体占据一定的体积，以促进转录、基因表达、DNA修复和重组等过程。尽管在过去的25年里，对细胞核的结构组织进行了大量的研究，但仍然完全不清楚染色体区域是如何在细胞分裂和细胞寿命期间建立和维持的。具体地说，关于这些区域是如何在有丝分裂后染色体去凝集后重新建立的知之甚少。端粒是组成真核线性染色体物理末端的核蛋白复合体，用于掩盖暴露的DNA，使其不被识别为双链断裂。人类端粒已经被证明定位于核的外围，特别是在有丝分裂后，当核重新组装时。因此，端粒锚定在包膜上可能是重组染色质结构域的一种可能方式。我将使用Hi-C，这是染色体构象捕获(3C)的扩展，随后是高通量测序，以公正、全基因组的方式确定端粒和基因组区域之间的相互作用。这将使我能够研究细胞核组织和远距离染色质相互作用在细胞周期中是如何变化的，并在细胞分裂后重新建立。此外，还将研究细胞老化过程中的变化和带有疾病突变的细胞。最后，我将阐述物理端粒锚定的分子机制，以便能够直接测试端粒锚定在确定基因组结构和组织中的作用。重要的是，区域的变化将是在衰老、细胞转化或疾病状态下改变全基因组转录签名和整个细胞核的有效方法。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地建立区域，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通路，最终目的是对抗这些通路中的病理变化，从而防止疾病的发生。

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.