Microscopy of histone-humanized yeast strains

Description

Eukaryotic DNA wraps around well-conserved histone octamers to form nucleosome fibers, which represents the most basic functional organization of chromatin among divergent organisms. As the effects of species-specific changes on nucleosome functions remain poorly characterized, histone replacement between divergent species can help identify processes that are functionally entrenched in different organisms. In this work we have characterized biological effects of the human-like nucleosomes, showing their direct impact on the overall structure of the chromatin in Saccharomyces cerevisiae. We used transmission electron microscopy (TEM) and found a size increase in the human nucleosome core particle (NCP), linked to shorter free linker DNA, suggesting that chromatin is less accessible. Moreover, human histones caused nucleolar fragmentation linked to rDNA array instability, through an RNA-polymerase II dependent mechanism, leading to its extraordinary, but entirely reversible, intra-chromosomal expansion.

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Transmission electron microscopy (TEM) for imaging chromatin fibers For the preparation of chromatin spreads in yeast we followed the published protocol described by (Osheim et al., 2008). We extracted and spread chromatin from log phase yeast cultures (Saccharomyces cerevisiae histones: BY4742; Homo sapiens histones: yDT92, yDT180) grown in YPD with 1M sorbitol at 30ºC. Approximately 10^7 cells were enzymatically lysed using a 1mg/ml Zymolyase 20T (US biological, Z1000) solution in YPD 1M sorbitol. Chromatin spreading was conducted in a 35 x 10 mm plastic petri dish containing a 0.025% Triton pH 9.1 solution that was incubated at room temperature and in mild agitation for 45 min. Spreading chromatin was mildly crosslinked with 1/10 [v/v] sucrose–formalin solution (100 mM sucrose, 3.7% formaldehyde Tousimis Researc Corporation, 1008A, with the pH adjusted to 8.8) for an additional ~30 min. Chromatin was deposited onto the EM carbon grids (Electron Microscopy Sciences, CF300-Cu) by centrifugation at 7000 x g (Centrifuge: Sorvall LXTR with swinging bucket rotor) for 10 min. Nucleic acid and protein staining were performed using 4% solutions of Uranyl Acetate (UA) (Electron Microscopy Sciences cat. 22400-4) and Phosphotungstic acid hydrate (PTA) (Sigma-Aldrich P4006-10G) in ethanol. Imagese were acquired using the electron transmission microscope (FEI Talos 120C TEM) at various resolutions, ranging from 10-kX to 150-kX, at the NYU Langone Microscopy Laboratory. Protein tagging and Fluorescent microscopy The organization of the nucleolus within the nucleus was monitored using fluorescently tagged proteins at their endogenous C-terminus. Nuclear envelope was labeled with mScarlet (NUP49::mScarlet-S.p. HIS5) and the nucleolus with GFP (NOP10::EGFP-KanMX) using reagents that we previously described in Lazar-Stefanita et al., 2023. Two independent isolates for each strain, containing either yeast (Sc) or human (Hs) histones, were validated for dual tagging based on their positive emission wavelengths in the GFP (513 nm) and RFP (605 nm) channels. The resulting strains (Sc: yLS110-C1 and yLS110-C3; Hs: yLS117-C1 and yLS117-C2) were grown in SC–His medium to saturation (24 h for yLS110 and 48 h for yLS117) and live cells were imaged in agarose pads prepared in SC–His medium (to prevent Brownian motion). Imaging was performed on the EVOS M7000 microscope using the Olympus X-APO 100 Oil, 1.45NA/WD 0.13mm (Oil) objective. Images were acquired as Z-stacks and visualized as max intensity projections using ImageJ (Schneider et al., 2012). Different fields of view were used to count nearly 1000 nuclei (496 for yLS110 and 477 for yLS117) displaying either one intact nucleolus or many fragmented nucleoli.

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