Gene dosage screens in yeast reveal core signalling pathways controlling heat adaptation

Abstract

Heat stress causes proteins to unfold and lose their function, jeopardizing essential cellular processes. To protect against heat and proteotoxic stress, cells mount a dedicated stress-protective programme, the so-called heat shock response (HSR). Our understanding of the mechanisms that regulate the HSR and their contributions to heat resistance and growth is incomplete. Here we employ CRISPRi/a to down- or upregulate protein kinases and transcription factors in S. cerevisiae. We measure gene functions by quantifying perturbation effects on HSR activity, thermotolerance, and cellular fitness at 23, 30 and 38°C. The integration of these phenotypes allowed us to identify core signalling pathways of heat adaptation and reveal novel functions for the high osmolarity glycerol, unfolded protein response and protein kinase A pathways in adjusting both thermotolerance and chaperone expression. We further provide evidence for unknown cross-talk of the HSR with the cell cycle-dependent kinase Cdc28, the primary regulator of cell cycle progression. Finally, we show that CRISPRi efficiency is temperature-dependent and that different phenotypes vary in their sensitivity to knock-down. In summary, our study quantifies regulatory gene functions in different aspects of heat adaptation and advances our understanding of how eukaryotic cells counteract proteotoxic and other heat-caused damage.

CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate

“…Guide RNA log2FCs are highly correlated with those of a previous study where the TF and PK libraries have been profiled individually with different transformation batches and without oxygen limitation (Jann et al, 2020), indicating high reproducibility of CRISPRi effects (Pearson R 2 =0.67, p-value<2.2e-16, Fig. 2c).…”

“…CRISPRi effects observed in one of the screens were explained by the other. Interestingly, we measured increased fitness for the repression of three genes (HAP1, RIM11 & RME1) that were not significant in the previous study (Jann et al, 2020), presumably due to the oxygenlimited conditions applied here to focus on fermentation. This is supported by the heme activated protein 1 (Hap1) TF which represses Rox1 in non-stress conditions (Zitomer and Lowry, 1992).…”

“…CRISPRi system has been deeply characterized (Smith et al, 2016(Smith et al, & 2017Jaffe et al, 2019;Jann et al, 2020), is freely available on addgene (#73796) and supported by an in-house developed gRNA design platform (http://lp2.github.io/yeast-crispri/; Smith et al, 2016) and a customizable computational analysis pipeline (Methods). The plasmid system can be transformed into any strain background, including polyploid and industrial strains, to devise strategies for improving process performance.…”

“…gRNA log2FC (Jann et al, 2020) gRNA log2FC (this study) screen from Jann et al, (2020). CRISPRi effects of both screens report on fitness in SC medium.…”

“…Accordingly, the ume6Δ mutant is sensitive to oxidative (Brown et al, 2006), hyperosmotic (Dudley et al, 2005) high temperature stress (Jann et al, 2020) and to more than 20 diverse chemicals some of which may have properties similar to lignocellulosic compounds (based on SGD; Cherry et al, 1998). 10 -3 10 -4 10 -5 10 -2 10 -3 10 -4 10 -5…”