Multiple molecular events underlie stochastic switching between 2 heritable cell states in fungi

Abstract

Eukaryotic transcriptional networks are often large and contain several levels of feedback regulation. Many of these networks have the ability to generate and maintain several distinct transcriptional states across multiple cell divisions and to switch between them. In certain instances, switching between cell states is stochastic, occurring in a small subset of cells of an isogenic population in a seemingly homogenous environment. Given the scarcity and unpredictability of switching in these cases, investigating the determining molecular events is challenging. White-opaque switching in the fungal species Candida albicans is an example of stably inherited cell states that are determined by a complex transcriptional network and can serve as an experimentally accessible model system to study characteristics important for stochastic cell fate switching in eukaryotes. In standard lab media, genetically identical cells maintain their cellular identity (either “white” or “opaque”) through thousands of cell divisions, and switching between the states is rare and stochastic. By isolating populations of white or opaque cells, previous studies have elucidated the many differences between the 2 stable cell states and identified a set of transcriptional regulators needed for cell type switching and maintenance of the 2 cell types. Yet, little is known about the molecular events that determine the rare, stochastic switching events that occur in single cells. We use microfluidics combined with fluorescent reporters to directly observe rare switching events between the white and opaque states. We investigate the stochastic nature of switching by beginning with white cells and monitoring the activation of Wor1, a master regulator and marker for the opaque state, in single cells and throughout cell pedigrees. Our results indicate that switching requires 2 stochastic steps; first an event occurs that predisposes a lineage of cells to switch. In the second step, some, but not all, of those predisposed cells rapidly express high levels of Wor1 and commit to the opaque state. To further understand the rapid rise in Wor1, we used a synthetic inducible system in Saccharomyces cerevisiae into which a controllable C. albicans Wor1 and a reporter for its transcriptional control region have been introduced. We document that Wor1 positive autoregulation is highly cooperative (Hill coefficient > 3), leading to rapid activation and producing an “all or none” rather than a graded response. Taken together, our results suggest that reaching a threshold level of a master regulator is sufficient to drive cell type switching in single cells and that an earlier molecular event increases the probability of reaching that threshold in certain small lineages of cells. Quantitative molecular analysis of the white-opaque circuit can serve as a model for the general understanding of complex circuits.

The transcription factor Ofi1 is critical for white‐opaque switching in natural MTLa/α isolates of Candida albicans

“…In turn, Wor2 activates the WOR1 expression, and Czf1 represses the EFG1 expression. Although it is known that concentrations of the four principal regulators must change during the white‐opaque switching event (Ziv et al., 2022), the mechanism by which the positive feedback loop is activated remains to be elucidated in C. albicans . In addition, transcriptional regulators Wor3 (Lohse et al., 2013) and Ahr1 (Wang et al., 2011) have been reported to be involved in the transcriptional network that specifies the white and opaque cell types (Hernday et al., 2013).…”