Transcriptional timing and noise of yeast cell cycle regulators—a single cell and single molecule approach

Gene expression is a stochastic process and its appropriate regulation is critical for cell cycle progression. Cellular stress response necessitates expression reprogramming and cell cycle arrest. While previous studies are mostly based on bulk experiments influenced by synchronization effects or lack temporal distribution, time-resolved methods on single cells are needed to understand eukaryotic cell cycle in context of noisy gene expression and external perturbations. Using smFISH, microscopy and morphological markers, we monitored mRNA abundances over cell cycle phases and calculated transcriptional noise for SIC1, CLN2, and CLB5, the main G1/S transition regulators in budding yeast. We employed mathematical modeling for in silico synchronization and for derivation of time-courses from single cell data. This approach disclosed detailed quantitative insights into transcriptional regulation with and without stress, not available from bulk experiments before. First, besides the main peak in G1 we found an upshift of CLN2 and CLB5 expression in late mitosis. Second, all three genes showed basal expression throughout cell cycle enlightening that transcription is not divided in on and off but rather in high and low phases. Finally, exposing cells to osmotic stress revealed different periods of transcriptional inhibition for CLN2 and CLB5 and the impact of stress on cell cycle phase duration. Combining experimental and computational approaches allowed us to precisely assess cell cycle progression timing, as well as gene expression dynamics.
Single-cell approach dissects noisy cell cycle transcription Single-molecule time-resolved mRNA quantification revealed long-term regulation of transcription and cell cycle progression upon osmotic stress. A team led by Edda Klipp at Humboldt-Universität zu Berlin, Germany, combined smRNA-FISH microscopy, genetic and morphological markers and stochastic modeling to monitor mRNA dynamics and related noise levels throughout cell cycle for SIC1, CLN2, and CLB5, main G1/S regulators in budding yeast. All genes were present throughout cell cycle enlightening that transcription is not divided in on and off but rather in high and low phases. Osmotic stress induced distinct periods of inhibition for CLN2 and CLB5, and long-term impact on cell cycle phase progression. This approach provided insights in transcription regulation unavailable with bulk experiments and chemical synchronization. Future research may address whether these patterns are general to other eukaryotic cell cycle regulators.