Characterization of topological machines that control chromosome conformation

项目概要/摘要 本研究的目的是研究染色体的分子机制 构象。染色体结构对于控制基因组过程（例如转录、 复制、染色体分离以及这种结构的破坏存在于遗传性疾病和 癌细胞。染色体组织主要分为三个层次：染色体区域、染色体 区室和拓扑关联域（TAD）。目前的模型是 TAD 形成 由于染色质纤维的动态环挤压，其被结合到的 CTCF 以定向方式阻断 TAD 边界。然而，这种拓扑机器的组成和分子机制 目前尚不清楚形成这些染色质环的因素。先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发挥作用。在 此外，最近的分子建模研究涉及环挤出，例如动态挤压DNA 环，作为染色体结构的一个重要特征，但这尚未经过测试 实验性地。本研究将结合Hi-C、ChIP-seq、TMP-seq等基因组方法 利用细胞生物学和功能遗传学方法来研究细胞的分子成分和动力学 拓扑机。为了解决这个问题，将执行三个互补的目标： 1) 评估角色 最近被鉴定为 TAD 边界 CTCF 复合体一部分的拓扑异构酶。 2） 研究修饰 TAD 生物学关键元件的组蛋白变体的作用。 3）开发新的 确定 TAD 内部染色质动力学的方法，以测试最近提出的 TAD 形成模型 动态环路形成。总之，完成这些目标将带来关于功能和功能的新见解。 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.