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

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 Surprisingly, the same chromatin organization is indicated by my, yet unpublished, recent measurements on Drosophila pericentric heterochromatin (PHC), thus suggesting a unified organization responsible for gene silencing.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