Temporal anatomy of an epigenetic switch in cell programming: the white-opaque transition of C. albicans

Abstract

The human pathogen Candida albicans undergoes a well-defined switch between two distinct cell types, named 'white' and 'opaque'. White and opaque cells differ in metabolic preferences, mating behaviours, cellular morphologies and host interactions. Each cell type is stable through many generations; switching between them is rare, stochastic and occurs without any known changes in the primary sequence of the genome; thus the switch is epigenetic. The white-opaque switch is regulated by a transcriptional circuit, composed of four regulators arranged in a series of interlocking feedback loops. To understand how switching occurs, we investigated the order of regulatory changes that occur during the switch from the opaque to the white cell type. Surprisingly, changes in key transcriptional regulators occur gradually, extending over several cell divisions with little cell-to-cell variation. Additional experiments, including perturbations to regulator concentrations, refine the signature of the commitment point. Transcriptome analysis reveals that opaque cells begin to globally resemble white cells well before they irreversibly commit to switching. We propose that these characteristics of the switching process permit C. albicans to 'test the waters' before making an all-or-none decision.

Figure 1

Working model of the white-opaque regulatory circuit and its activity in white and opaque cells. (a) In white cells, EFG1 represses WOR1 indirectly through WOR2 to maintain white cell identity. (b) In opaque cells, WOR1, WOR2, and CZF1 establish a series of positive feedback loops, maintaining opaque cell identity and repressing EFG1. Up-regulated genes and active relationships are indicated in black. Down-regulated genes are indicated in gray. Arrows and bars represent activation and repression respectively. Figure adapted from Zordan et al., 2007 (Zordan et al., 2007).

Figure 2

Changes in protein levels of Wor1, Wor2, Czf1, and Efg1 during the opaque to white switch. (a) Commitment point. Opaque cells in suspended culture were shifted from 25°C to 37°C at T=0 hr. At each time point, an aliquot of cells was collected and plated at room temperature to determine the percentage of cells that had switched to the white cell type. Data points reflect the mean of six experiments (except for the 9 hour time point which reflects 5) and error bars represent the standard error of the mean. (b) Levels of the four regulatory proteins as determined by western blotting. As described in the methods, protein levels were normalized to the maximum levels for each regulator. For Wor1, Wor2, and Czf1 the maximum level occurs in opaque cells, while Efg1 is maximally expressed in white cells. Data points reflect the mean of four (Wor1 and Efg1) or three (Wor2 and Czf1) experiments; error bars for each protein level are shown in Supplemental Figure S1. (c) Commitment point for the experiments in (d) and (e); the percentage of colonies with the white colony phenotype is plotted for wild type, Wor1-GFP, and Efg1-GFP containing strains. (d) Single cell measurements of Wor1. Wor1-GFP levels in individual cells were quantitated as described in the methods. The red line represents the median value at each time point and each dot represents a single cell. (e) Single cell measurements of Efg1; symbols are as described in (d).

Figure 3

Transcript levels of WOR1, WOR2, CZF1, and EFG1. (a) Microarray transcript data, with the median of four independent experiments shown. The first block represents changes in opaque cells during the time course, normalized to the starting (T=0 hr) opaque cells. The second block highlights the changes due to temperature alone by comparing a time course performed on white cells (which do not switch) to the starting (T=0 hr) culture of white cells. The last block shows the profile of the opaque cell cultures at each time point, compared to the white cell cultures at the same time point. The color key is based on a log2 scale. (b) Levels of WOR1, WOR2, and EFG1 transcripts as determined by RT-qPCR (left axis). The levels of each transcript are normalized to their maximum levels in opaque (WOR1, WOR2) or white (EFG1) cells. The right axis displays the percentage of colonies with the white colony phenotype for the same experiment.

Figure 4

Behavior of 209 white- and opaque-enriched genes over the time course of switching. The three column blocks are described in Figure 3a, with the median value from the four experiments shown for each gene. (a) Changes in the 145 opaque-enriched genes during the time course. (b) Changes in the 64 white-enriched genes during the time course. The color key for (a) and (b) is based on a log2 scale. (c) The six gene sets were binned and the mean transcriptional change at each time point for each set was determined in opaque cells. Sets of genes (1, 2, 3, etc.) are described in more detail in the text. Sets 1 and 2 are the opaque-enriched genes that are rapidly down-regulated upon the temperature shift. Set 3 is the 11 gene WOR1/CZF1 cluster of opaque-enriched genes. Set 4 contains the white-enriched “early” genes. Set 5 is white-enriched “middle” genes, and Set 6 is the white-enriched “late” gene group. Data are from the same set of experiments shown Figure 3a.

Figure 5

Effects of deleting one copy of WOR1, WOR2, CZF1, or EFG1 on the commitment point. (a) Temperature-induced switching from opaque to white cell types for the wild type strain and for heterozygous deletions of the four known regulators. The percentage of colonies with the white colony phenotype is plotted for each strain. The 50% commitment points for these strains are 5 hours (wild type and CZF1 het), 3 hours (WOR1 het), and 6 hours (EFG1 het). As the WOR2 heterozygous strain had a consistent background of 20–30% white cells, we have defined the commitment point for this strain as the point at which half the opaque colonies, or 60–65% of the total colonies, are white (approximately 4 hours). All temperature shift experiments in (a) were performed in parallel on the same day. (b) Wor1 protein levels in wild type, WOR1 heterozygous, and EFG1 heterozygous strains. Western blotting data (left axis, dashed lines) and percent commitment (right axis, solid lines) are plotted for Wor1 in wild type (red), WOR1 heterozygous (green), and EFG1 heterozygous (blue) strains. Data points represent the mean of two experiments.

Figure 6

Order and timing of events following temperature shift of an opaque population.