Mapping the short-range chromatin architecture of the repressive epigenome

项目摘要 基因组结构与许多重要的细胞过程有关，从转录调控到 染色体分离。最近的技术创新使人们能够详细地描述长期的 范围染色体构象。在抑制区出现远距离的染色体紧密连接 统称为异染色质。这些基因组区域对于适当的细胞类型依赖至关重要 基因表达模式和它们的结构也有助于防止基因组的不稳定性 寄生转座子的表达，并通过调节着丝粒附近的染色质结构， 端粒和其他DNA重复序列。尽管在理解远距离染色质压缩方面取得了进展，但很少有 存在着在亚核小体分辨率下测量DNA的空间组织的方法，即长度 与转录和其他关键DNA过程相关的标度。此外，许多异色结构 含有DNA重复序列，由于它们无法被映射到单个基因组位置，因此很难研究。 我试图使用最近开发的一种方法来确定异染色质的短程压缩状态， RICC-SEQ，它可以在亚核小体分辨率下测量3D DNA接触。我将创建新的RICC-seq- 基于纳米孔长读测序的方法，使DNA重复测量成为可能。我也会 通过基因操作调节异染色质的组蛋白修饰途径来确定它们的影响 关于短程染色质结构。组蛋白去乙酰化和甲基化是表观遗传的两条主要途径 来动态调节异染色质。除了这些修饰外，该基因的多种亚型 保守的异染色质蛋白1(HP1)有助于调节异染色质结构。我会决定 这些表观遗传因子在体内对染色质压缩和转录的各自贡献 沉默。除了定义管理异染色质组织和功能的基本规则外，我还 建议研究相分离凝析油的压缩状态。相分离被认为是 调节异染色质动力学和转录，然而，它如何影响短距离染色质 组织问题尚未得到解决。我将测定体外相的3D DNA折叠构象- 染色质的分离，体外观察到的现象与染色质紧凑的测量 细胞。这项拟议的工作将梳理出纳米级基因组组织的基本原理 为理解异染色质调控及其可能的影响提供了结构基础 它对疾病状态的破坏。

Project Summary Genome architecture is associated with many essential cellular processes from transcriptional regulation to chromosome segregation. Recent technological innovations have enabled detailed characterization of long- range chromosome conformations. Long-range chromosome compaction appears at repressive regions collectively referred to as heterochromatin. These genomic regions are vital for proper cell type-dependent gene expression patterns, and their architecture also helps to protect against genomic instability by controlling the expression of parasitic transposons and by regulating the chromatin structure near centromeres, telomeres, and other DNA repeats. Despite advances in understanding long-range chromatin compaction, few methods exist that measure the spatial organization of DNA at sub-nucleosome resolution, which is the length scale relevant to transcription and other critical DNA processes. Furthermore, many heterochromatic structures contain DNA repeats, which are difficult to study due to their inability to be mapped to a single genomic locus. I seek to determine the short-range compaction states of heterochromatin, using a recently developed method, RICC-seq, which can measure 3D DNA contacts at sub-nucleosome resolution. I will create new RICC-seq- based methods using Nanopore long-read sequencing to enable measurements of DNA repeats. I will also genetically manipulate histone modification pathways that regulate heterochromatin to determine their effects on short-range chromatin structure. Histone deacetylation and methylation are two major epigenetic pathways that dynamically regulate heterochromatin. In addition to these modifications, multiple isoforms of the conserved heterochromatin protein 1 (HP1) help regulate heterochromatin structures. I will determine the respective in vivo contributions that these epigenetic factors have on chromatin compaction and transcriptional silencing. In addition to defining the basic rules governing heterochromatin organization and function, I also propose to investigate the compaction states of phase-separated condensates. Phase separation is thought to regulate heterochromatin dynamics and transcription, however, how it affects short-range chromatin organization has yet to be addressed. I will determine the 3D DNA folding conformations of in vitro phase- separated chromatin, connecting phenomena observed in vitro with measurements of chromatin compaction in cells. This proposed work will tease out fundamental principles of genomic organization at nanoscale resolution and provide a structural foundation for understanding heterochromatin regulation and the possible impacts of its disruption in disease states.