Characterization of topological machines that control chromosome conformation

项目概要/摘要 本研究的目的是探讨染色体的分子机制， 构象染色体结构对于控制基因组过程如转录， 复制、染色体分离和这种结构的破坏存在于遗传疾病中， 癌细胞染色体的组织结构主要有三个层次：染色体区域、染色体 区室和拓扑关联域（TADs）。目前的模型是， 由于染色质纤维的动态环挤出，其被CTCF以定向方式阻断， 界限。然而，这种拓扑机器的组成部分和分子机制 形成这些染色质环的是目前未知的。以前的研究表明，拓扑异构酶和 组蛋白变体可能在调节染色体构象和拓扑机器活性中起作用。在 此外，最近的分子建模研究已经涉及环挤出，例如动态挤出的DNA 环，作为染色体结构的一个重要特征，然而这还没有得到验证 实验性的本研究将结合使用Hi-C、ChIP-seq和TMP-seq等基因组方法 用细胞生物学和功能遗传学的方法来研究细胞的分子组成和动力学， 拓扑机为解决这一问题，将实现三个互补目标：1）评估 拓扑异构酶，最近已被确定为一部分的CTCF复杂的边界。（二） 研究组蛋白变异体的作用，这些变异体修饰了生物学中涉及的关键元素。3)开发新 方法来确定TADs内的染色质动力学，以测试最近提出的染色质形成模型， 动态回路形成总之，这些目标的完成将导致对功能的新见解， 染色体构象的调节。

PROJECT SUMMARY/ABSTRACT The goal of this research is to investigate the molecular mechanisms responsible for chromosome conformation. Chromosome structure is important for controlling genomic processes such as transcription, replication, and chromosome segregation, and disruptions to this structure are found in genetic diseases and cancer cells. There are three main levels of chromosome organization: chromosome territories, chromosome compartments, and topologically associating domains (TADs). The current model is that TAD formation occurs due to dynamic loop extrusion of chromatin fibers, which is blocked in a directional manner by CTCF bound to TAD boundaries. However, the components and molecular mechanism of this proposed topological machine which forms these chromatin loops are currently unknown. Previous studies suggest that topoisomerases and histone variants may have a role in regulating chromosome conformation and topological machine activity. In addition, recent molecular modeling research has implicated loop extrusion e.g. dynamically extruded DNA loops, as an important characteristic of chromosome structure, however this has not yet been tested experimentally. This study will use genomic methods such as Hi-C, ChIP-seq, and TMP-seq in combination with cell biological and functional genetic approaches to study the molecular components and dynamics of the topological machine. Three complementary aims will be performed to address this question: 1) Assess the role of topoisomerases that have recently been identified as a part of the CTCF complex at TAD boundaries. 2) Investigate the role of histone variants that decorate key elements involved in TAD biology. 3) Develop new methods to determine chromatin dynamics inside TADs to test recently proposed models of TAD formation by dynamic loop formation. Together, completion of these aims will lead to new insights about the function and regulation of chromosome conformation.