Heterogeneous non-canonical nucleosomes predominate in yeast cellsin situ

Abstract

Nuclear processes depend heavily on the organization of chromatin, whose subunits are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ resemble the canonical structure determined in vitro 24 years ago. The structure of nucleosomes in situ is otherwise poorly understood. Here we use cryo-ET and 3-D classification analysis to study the structure of yeast nucleosomes both in vitro and in situ. We show that the class averages of GFP-tagged yeast nucleosomes in vitro resemble canonical nucleosomes, with additional GFP densities. In contrast, none of the class averages of nucleosome-like particles in situ (inside cells) resemble canonical nucleosomes. The heterogeneous nature of the in situ class averages suggests that the intranuclear environment favors multiple conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average yeast nucleosome's DNA is partially detached in situ.

Nucleosome plasticity is a critical element of chromatin liquid–liquid phase separation and multivalent nucleosome interactions

“…Our work focuses on assessing an important feature of nucleosomes that likely pertains to chromatin LLPS: their inherent plasticity. That is, rather than static building blocks-as routinely considered in large-scale chromatin structural modelsat short length-scales nucleosomes are highly dynamic and structurally irregular entities 4,[61][62][63][64][65][66] , and are better described as a "dynamic family of particles" 67 . Nucleosomes in vivo can pack a broad range of DNA base pairs around the histone core (~100-170 bp), and can also have varied histone compositions and stoichiometries 67 .…”

Genome wide nucleosome landscape shapes 3D chromatin organization

“…Broadening of nucleosomal peaks is observed in vitro as compared to in vivo (fig. S9, A and B), possibly due to some shifting of nucleosomes during chromatin purification or due to differential breathing of DNA wrapped around histone octamer in in vitro versus in situ condition ( 29 ). Under in vitro conditions, averaged nucleosome profile around TSS of genes shows that peaks corresponding to downstream nucleosomes (from +2) are relatively better resolved with respect to +1 nucleosome in WT but merged in TKO (fig.…”

“…Well-positioned nucleosomes of the WT strain interact with each other establishing short-range contacts. In the case of the TKO strain, most of the nucleosomes are closely packed with a smaller fraction having longer spacing ( 29 ). In TKO strain, fewer short-range contacts are formed.…”

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

“…Then this additional knowledge can help guide localization during cryo-FIB-milling, cryo-ET collection, and cryo-ET processing. Additional labels for protein localization in tomograms, including metal and nonmetal tags (Silvester et al, 2021;Tan et al, 2021), may be biochemically added to the labeled specimen to help identify and understand the morphologies of the structures of interest in the corresponding label-free specimen.…”

“…Perhaps the most promising development in SRLM is MINFLUX, which may localize proteins with 1-3 nm precision (Gwosch et al, 2020) if devitrification issues with frozen specimens are first overcome. Multiple non-metal EM tags for labeling and identifying proteins of interest vitrified in situ have been proposed (Silvester et al, 2021;Tan et al, 2021), yet are not in mainstream use and may disrupt native protein conditions. In this review, we focus on label-free MS approaches for detecting protein quantities, their modifications, protein-protein interactions (PPIs), and protein complex components in conjunction with cryo-EM/ET.…”