Identification of the structural basis of heterochromatin-mediated gene silencing using correlative cryo-light and electron microscopy

使用相关冷冻光和电子显微镜鉴定异染色质介导的基因沉默的结构基础

• 批准号: 289147632
• 负责人: Dr. Mikhail Eltsov, Ph.D.
• 金额: --
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/289147632?language=en
• 关键词: Identification; structural; basis; heterochromatin; mediated

Cellular differentiation requires a controllable inactivation of a large part of the cellular genome. It begins with epigenetic modifications at the nucleosome level, which initiates DNA rearrangement into a compact state, incompatible with transcription, designated as heterochromatin. Heterochromatin formation is based on the binding of chromodomain proteins such as heterochromatin protein 1 (HP1) to modified nucleosomes, leading to their arrangement into higher-order structures. Although fundamental to understand global gene regulation, heterochromatin organization is poorly understood at the structural level. In vitro studies have so far revealed two types of possible multi-nucleosome arrangements, the single-start solenoid and the two-start zigzag helix. However, the higher order chromatin organization responsible for gene silencing inside the cell remains completely unclear, because of the difficulty to preserve the native chromatin state and the lack of structure determination methods with sufficient resolution for in situ specimen analysis. I aim to overcome these problems by combining live cell light microscopy with cryo-electron tomography (cryo-ET), a technique that has undergone a resolution revolution enabling 3D visualization of macromolecular complexes at nanometer resolution in situ within the cell. In my previous work, I discovered a conserved two-start arrangement of 30 nm chromatin fibers in transcriptionally inactive avian nuclei. Surprisingly, the same chromatin organization is indicated by my, yet unpublished, recent measurements on Drosophila pericentric heterochromatin (PHC), thus suggesting a unified organization responsible for gene silencing. Made feasible by the massively improved resolution provided by direct electron detectors and novel data analysis algorithms, I now plan to resolve the 3D path of DNA fibers in situ to directly determine the structural basis of transcriptional inaccessibility. Based on the normal heterochromatin structure, I will subsequently address the molecular basis of fiber formation, by both determining the heterochromatin structure in HP1 mutants as well as after full or partial depletion of HP1 using in vivo RNA interference systems. This project will resolve the physiological structure of heterochromatin under native conditions, thereby providing the structural basis to understand epigenetic gene silencing and the role of heterochromatin proteins in its establishment.

细胞分化需要大部分细胞基因组的可控失活。它开始于核小体水平的表观遗传修饰，其启动DNA重排成与转录不相容的紧凑状态，称为异染色质。异染色质的形成是基于染色质结构域蛋白如异染色质蛋白1（HP 1）与修饰的核小体的结合，导致它们排列成更高级的结构。尽管异染色质组织对于理解整体基因调控至关重要，但在结构水平上了解甚少。迄今为止，体外研究已经揭示了两种类型的可能的多核小体排列，单起始螺线管和双起始之字形螺旋。然而，负责细胞内基因沉默的高阶染色质组织仍然完全不清楚，因为难以保存天然染色质状态和缺乏足够分辨率的结构测定方法用于原位标本分析。我的目标是克服这些问题相结合的活细胞光学显微镜与冷冻电子断层扫描（cryo-ET），一种技术，已经经历了分辨率革命，使三维可视化的大分子复合物在纳米分辨率原位细胞内。在我以前的工作中，我发现了一个保守的两个开始安排的30纳米染色质纤维在转录不活跃的鸟类细胞核。令人惊讶的是，同样的染色质组织表明，我，但未发表，最近的测量果蝇臂间异染色质（PHC），从而表明一个统一的组织负责基因沉默。由于直接电子探测器和新型数据分析算法提供的大幅提高的分辨率，我现在计划原位解析DNA纤维的3D路径，以直接确定转录不可达性的结构基础。基于正常的异染色质结构，我将随后解决纤维形成的分子基础，通过确定异染色质结构在HP 1突变体以及后完全或部分消耗HP 1使用体内RNA干扰系统。该项目将解析异染色质在天然条件下的生理结构，从而为理解表观遗传基因沉默及其建立中异染色质蛋白的作用提供结构基础。