4D reorganization of yeast megachromsomes unveils spatio-temporal aspects of replication firing and gene contacts in yeast
Eukaryotic genomes vary greatly in terms of size, chromosome number and genetic complexity. Here we used engineered karyotypes of budding yeast – in which 16 native chromosomes were fused into two gigantic DNA molecules - to characterize effects of chromosome length on nuclear architecture and function. We reasoned that a ~5-fold increase in chromosome length - relative to the longest native chromosome - might alter nuclear occupancy of the megachromosomes. To investigate this hypothesis, we measured the proportion of the nucleus occupied by genomic DNA in cells labeled with SYTOX Green (see methods below). We observed that megachromosomes occupy a larger fraction of nuclear volume than native chromosomes that was correlated with an increase in the size of the nucleus.
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DNA staining with SYTOX Green: Approximately 10^7 cells/sample were fixed in 70% ethanol and stored at 4°C overnight. Cell pellets were washed twice with RNAse solution (10 mM Tris pH 8.0, 15 mM NaCl) and treated with 0.1 mg/ml RNAse A for 3-4 h at 37 ̊C. Cells were centrifuged, washed once with 50 mM Tris pH 7.5 and resuspended in labeling solution (1 μM SYTOX Green in 50 mM Tris pH 7.5; ThermoFisher) for 1 h at 4 ̊C. Before imaging and flow cytometry data acquisition, cells were washed twice and resuspended in 50 mM Tris pH 7.5. Imaging and estimation of DNA occupancy: To minimize the variability introduced by experimental procedure, the surface area occupied by the DNA was calculated in groups of samples manipulated simultaneously (on the same day). Exponentially-growing cultures were treated for DNA staining (see above). For Z-stack acquisition, concanavalin A coated glass slides were used to immobilize cells. Imaging was performed using the total internal reflection fluorescence (TIRF) microscope (objective: Plan Apo VC 100x Oil DIC N2; camera: Andor Zyla VSC-03679). NIS element AR software was used for image acquisition: 470 nm excitation wavelength (3% power), 10 ms exposure time, z-stack 1.8 μm range (relative positions in Z -0.9 to +0.9 μm, 7 steps of 0.3 μm). Images were imported and analyzed with Fiji software. The surface area occupied by the DNA was estimated using the “3D object counter” option after image segmentation and thresholding set at 3500-pixel intensity Supplemental Table 2a. Approximately 300 – 500 DNA surfaces/strain were plotted using the violin function and the p-values were calculated using the Kolmogorov–Smirnov test (K–S test) functions available in MatLab R2018. The increase in DNA occupancy in the size-matched n=2 strain was correlated with an enlargement of the nuclear size in this latter compared to the wild type, n=16. The surface area of the nucleus was measured in n=2 (LS126) and n=16 (LS125) yeasts, in which the nuclear envelope protein, Nup49, was tagged with mScarlet. Live cells in exponential growing phase were imaged using the EVOS M7000 microscope (Olympus X-APO 100X Oil, 1.45NA/WD 0.13mm oil objective). Images were analyzed with Fiji software. Spherical nuclei (~150 for n=16; ~300 for n=2) were manually selected and their circumference and surface area were estimated Supplemental Table 2b. Experiments were performed on two and three independent clones of LS125 and LS126.