Gene dosage and protein valency impact phase separation and fungal cell fate
Collin Ganser, Peiling He, Corey Frazer, Damian J. Krysan, Richard J. Bennett
Abstract
Cell fate decisions in eukaryotes are regulated by interconnected networks of transcription factors (TFs) that drive heritable changes in identity. However, much is unknown about how TFs act together to control cell fate, despite links to cellular dysfunction and disease when TF function is aberrant. Here, we addressed the interplay between TFs that control heritable switching in the diploid fungal pathogen Candida albicans. This species can propagate in two distinct cell states, white and opaque, with epigenetic transitions between states regulated by a core network of eight TFs plus >100 auxiliary TFs.
Introduction
Transcription factor (TF) networks play a central role in regulating cell fate across the tree of life, from bacteria to multicellular eukaryotes [1–3]. In microbial systems, transitions between alternative cell states can promote bet hedging to promote survival under fluctuating environmental conditions or can enable a division of labor between specialized cell types [4–6]. In higher organisms, cell fate transitions are associated with cell differentiation during development, cell reprogramming to generate pluripotent stem cells, and the formation of aberrant cell types during tumorigenesis [7,8].
Materials and method
Media
C. albicans strains were maintained on yeast peptone dextrose (YPD) agar plates, synthetic complete dextrose (SCD) agar plates [85], or CHROMagar plates (Fisher Scientific, NC9514149). Variations of SCD containing different amounts of glucose and N-acetylglucosamine (GlcNAc) were used for switching assays including SCD with 2% glucose, SCD with 1% glucose and 1% GlcNAc, and SCD with 1.75% glucose and 0.25% GlcNAc. All SCD variations used in switching assays were supplemented with 5 μg/mL Phloxine B to stain opaque colonies/sectors on agar plates [86].
Results
Analysis of heterozygous TF deletions for white-opaque switching
Genetic interactions between the core white-opaque TFs were examined using both simple and complex haploinsufficiency (CHI) analyses, in which C. albicans strains were heterozygous for one or two genes, respectively. Switching frequencies were evaluated in MTLa/a or MTLa/Δ derivatives of SC5314 MTLa/α cells, as a/α cells are restricted from undergoing switching due to inhibition via MTLa1/α2 [52–54].
Discussion
The C. albicans white-to-opaque switch represents an exemplary system to dissect the transcriptional control of epigenetic cell fate. This switch is controlled by a core network of eight TFs that are potentially recruited to the DNA by their coalescence into biomolecular condensates [36,43,49]. However, despite the identification of >100 TFs whose deletion impacts white-opaque switching [49,58], there remains limited understanding of how these TFs act in combination to regulate the switch. This is in part due to several TFs being essential to switching, with cells lacking WOR1 or WOR4 being locked in the white state whereas those lacking RBF1 or SSN6 are locked in the opaque state [37,39–42,58].
Acknowledgments
We thank Alexander Johnson (UCSF), Aaron Hernday (UC Merced), Steven Sandler, UMass Amherst) and Joseph Heitman (Duke University) for plasmids and strains, and Robert Tjian (UC Berkeley) for U2OS cells.
Citation: Ganser C, He P, Frazer C, Krysan DJ, Bennett RJ (2025) Gene dosage and protein valency impact phase separation and fungal cell fate. PLoS Genet 21(8): e1011810. https://doi.org/10.1371/journal.pgen.1011810
Editor: David Kadosh, UT Health San Antonio: The University of Texas Health Science Center at San Antonio, UNITED STATES OF AMERICA
Received: October 18, 2024; Accepted: July 17, 2025; Published: August 8, 2025
Copyright: © 2025 Ganser et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are included within the manuscript and its Supporting Information files.
Funding: This work was supported by NIH/NIAID awards AI081704 and AI141893 to RJB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
