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

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，它启动DNA重排成致密状态，与转录不相容，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 (HP1)）与修饰核小体的结合，导致它们排列成高阶结构。虽然对理解全球基因调控至关重要，但在结构水平上对异染色质组织的了解甚少。到目前为止，体外研究已经揭示了两种可能的多核小体排列，单启动螺线管和双启动之字形螺旋。然而，细胞内负责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以及缺乏具有足够分辨率的原位标本分析的结构测定方法。我的目标是通过将活细胞光显微镜与低温电子断层扫描（cryo-ET）相结合来克服这些问题，低温电子断层扫描（cryo-ET）是一种经历了分辨率革命的技术，能够在细胞内原位以纳米分辨率进行大分子复合物的3D可视化。在我之前的工作中，我在转录不活跃的鸟类细胞核中发现了30 nm染色质纤维的保守双启动排列。令人惊讶的是，我最近对果蝇的外中心异染色质（PHC）的测量表明了相同的染色质组织，但尚未发表，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分辨率大幅提高，我现在计划在原位解析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，我随后将通过确定HP1突变体中的异染色质结构以及使用体内RNA干扰系统完全或部分耗尽HP1后的异染色质结构来解决纤维形成的分子基础。本项目将解析天然条件下异染色质的生理结构，从而为理解表观遗传基因沉默和异染色质蛋白在其建立中的作用提供结构基础。

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 Surprisingly, the same chromatin organization is indicated by my, yet unpublished, recent measurements on Drosophila pericentric heterochromatin (PHC), thus suggesting a unified organization responsible for gene silencing.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.