NAT-resistant transformants were PCR tested for correct integration of the cassette as described for CaLC2034

NAT-resistant transformants were PCR tested for correct integration of the cassette as described for CaLC2034. transcription 2-Hydroxybenzyl alcohol factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit -1,3-glucan synthesis. Cas5 has distinct transcriptional 2-Hydroxybenzyl alcohol targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance. Introduction The fungal kingdom encompasses diverse species, including a minority that have a devastating impact on human health. One of the most pervasive fungal pathogens of humans is usually can exploit a decline in host immunity or an imbalance in the host microbiome, leading to diverse pathologies such as oral thrush, vaginal candidiasis, or life-threatening bloodstream infections with mortality rates of ~40%4, 5. thrives as a human pathogen in part due to its ability to evade host immunity by switching between yeast and filamentous morphologies, as well as due to its capacity to withstand the hostile host environment by activating strong stress responses6. The emerging paradigm is usually that stress response pathways are not only critical for adaptation to host conditions, but they also enable fungal virulence and drug resistance7C11. The emergence of resistance to the limited arsenal of antifungal drugs impedes the effective treatment of systemic infections12C14. A poignant example is the evolution of resistance to the only new class of antifungal to be approved in decades, the echinocandins15, 16. Echinocandins block -1,3-glucan biosynthesis in the fungal cell wall via inhibition of the glucan synthase Fks1, thereby compromising cell wall integrity. The most common mechanism of echinocandin resistance involves mutations in the drug target mobilizes diverse stress response programs through the action of transcription factors. For example, in response to cell membrane and cell wall stress, the transcription factor Crz1 is activated by calcineurin, leading to the induction of calcineurin-dependent genes19, 20. Another example from the model HDAC3 yeast is the cell wall stress-dependent activation of the transcription factor Rlm1 by the MAP kinase Mpk121. Although Rlm1 is the main transcriptional 2-Hydroxybenzyl alcohol regulator of cell wall stress responses in and most other eukaryotes, and the mechanism by which it is regulated remains enigmatic. Activation of stress responses can induce diverse physiological changes, including modulation of cell cycle progression and remodeling of cell wall architecture23C27. The most well characterized stress response pathway involved in cell cycle regulation is controlled by the MAP kinase Hog126. In response to osmotic stress, Hog1 mediates a transient cell cycle arrest to enable cellular adaptation26. Multiple stress response pathways coordinate cell wall remodeling in response to environmental perturbations, including heat shock27, osmotic stress28, and cell wall stress29. However, little is known about whether cell cycle progression and cell wall remodeling are coordinated in response to stress in were significantly enriched in genes with functions in diverse processes, including metabolic processes and conversation with host (Fig.?1a and Supplementary Data?1). In contrast, the gene set that had increased RNA PolII occupancy in a mutant relative to wildtype. Enriched GO processes are indicated, and were clustered using the DAVID Gene Functional Classification Tool. b Bar chart showing the number of genes differentially bound by PolII (differentially bound genes), with increased binding in and decreased binding in and decreased binding in and decreased binding in mutant, revealed that >60% of caspofungin-responsive genes were 2-Hydroxybenzyl alcohol dependent on Cas5. Specifically, 163 of the 294 genes with increased PolII occupancy in response to caspofungin exposure and 178 of the 252 genes with reduced occupancy were dependent on Cas5 (Fig.?1f and Supplementary Data?2C4). These findings suggest that Cas5 has a profound impact on global transcriptional responses to cell wall stress. Finally, we focused on those genes with Cas5-dependent differences in RNA PolII binding under basal and cell wall stress conditions. Strikingly, only 28% of genes with Cas5-dependent differences in RNA PolII binding were common to both untreated and caspofungin treatment conditions (Fig.?2a and Supplementary Data?1, 3 and 4). The Cas5-dependent genes specific to each condition had.