Epigenetic properties of white-opaque switching in Candida albicans are based on a self-sustaining transcriptional feedback loop

Abstract

White-opaque switching in the human fungal pathogen Candida albicans is an alternation between two distinct types of cells, white and opaque. White and opaque cells differ in their appearance under the microscope, the genes they express, their mating behaviors, and the host tissues for which they are best suited. Each state is heritable for many generations, and switching between states occurs stochastically, at low frequency. In this article, we identify a master regulator of white-opaque switching (Wor1), and we show that this protein is a transcriptional regulator that is needed to both establish and maintain the opaque state. We show that in opaque cells, Wor1 forms a positive feedback loop: It binds its own DNA regulatory region and activates its own transcription leading to the accumulation of high levels of Wor1. We further show that this feedback loop is self-sustaining: Once activated, it persists for many generations. We propose that this Wor1 feedback loop accounts, at least in part, for the heritability of the opaque state. In contrast, white cells (and their descendents) lack appreciable levels of Wor1, and the feedback loop remains inactive. Thus, this simple model can account for both the heritability of the white and opaque states and the stochastic nature of the switching between them.

Fig. 1.

Alignment of Wor1 homologs across fungal species. Protein sequences were aligned by using ClustalW (15), which identified a highly conserved region at the N terminus of each protein (blue). For instance, C. albicans Wor1 and Sc. pombe Gti1 are 53% identical across the conserved region. C. albicans Wor1 protein is 785 aa in length, and the other proteins are drawn to scale.

Fig. 2.

Ectopic expression of WOR1 in white cells drives the cells to the opaque phase. (A and B) Ectopic expression of WOR1 causes white cells to resemble opaque cells in appearance. Cell dimensions were measured in differential interference contrast images (B), and populations of cells were compared based on the distribution of length/width ratios for 50 cells per strain for each condition (A). (C) Ectopic expression of WOR1 in white cells causes them to express genes characteristic of opaque cells. Quantitative RT-PCR was used to monitor transcription of the white-specific genes WH11 and EFG1 and the opaque-specific genes SAP1 and OP4. All values were normalized to PAT1, a transcript that is not regulated by white–opaque switching. (D) Ectopic expression of WOR1 in white cells renders them sensitive to the mating pheromone α-factor. This specialized property of true opaque cells is visualized by the formation of mating projections on the ends of the cells (14). Cells were treated with α-factor (10 μg/ml in DMSO) or an equivalent amount of DMSO as a control. In A and B, “ON” and “OFF” indicate the expression of the pMET3-WOR1 construct, as controlled by media conditions. For strains that lack the pMET3-WOR1 construct, media conditions are designated by “ON” or “OFF.” For experiments shown in C and D, strains were grown in media that induces pMET3-WOR1 expression. All strains are a strains.

Fig. 3.

Northern and Western blot analysis of WOR1 expression. (A) Northern blot analysis of total RNA isolated from strains grown under conditions that induce the pMET3-WOR1 construct. The relevant genotypes are indicated above each lane (all strains are a strains). RPL5 serves as a loading control. (B) Immunoblot analysis of Wor1 protein levels in white and opaque cells of several different strains. Wor1 was detected in WCE by using an antibody (α-Wor1) generated against a peptide portion of Wor1. Strain 1 (a, wor1Δ) controls for nonspecific binding of the antibody. Strain 2 (CAF2-1, a, white) and strain 3 (CAF2-1, a, opaque) show the differential expression of Wor1 between white and opaque cells. Strain 4 (a, pMET3-WOR1), strain 5 (a, wor1Δ + pMET3-WOR1), and strain 6 (a/α, pMET3-WOR1) show that the pMET3-WOR1 construct is tightly regulated by media conditions and that the protein is not grossly overexpressed. Strain 7 (SN87, a, white) and strain 8 (SN87, a, opaque) again show the differential expression of Wor1 in white versus opaque cells. The blot was stripped and reprobed with α-Tub1 as a loading control. “ON” and “OFF” indicate media conditions used to regulate the pMET3-WOR1 construct, as described in Fig. 2.

Fig. 4.

Wor1 protein is bound to the region upstream of its gene. ChIP was performed with α-Wor1 antibodies in wild-type a opaque, wild-type a white, wild-type a/α, and wor1Δ a strains. Wor1 ChIP enrichment was detected by quantitative PCR at ≈250-bp intervals across the 10.3-kb intergenic region. Shown are enrichment values at each position upstream of WOR1 relative to a control gene (ADE2) that is not regulated by white–opaque switching.

Fig. 5.

Ectopic expression of WOR1 in a/α cells induces opaque-like characteristics. Ectopic expression of WOR1 was regulated by using the pMET3-WOR1 construct in a/α strains. (A) Ectopic expression of WOR1 causes white cells to resemble opaque cells in appearance. Cell dimensions were measured in differential interference contrast images, and populations of cells were compared based on the distribution of length/width ratios for 50 cells per strain for each condition. “ON” and “OFF” indicate media conditions used to regulate the pMET3-WOR1 construct, as described in Fig. 2. (B) Ectopic expression of WOR1 in white cells causes them to express genes characteristic of opaque cells. Quantitative RT-PCR was used to monitor transcription of the white-specific genes WH11 and EFG1 and the opaque-specific genes SAP1 and OP4 under conditions that induce the pMET3-WOR1 construct. All values were normalized to PAT1, a transcript that is not regulated by white–opaque switching.

Fig. 6.

A model for a positive Wor1 feedback loop in regulation of white–opaque switching.