Chien-Der Lee,Naomi Ziv,Alexander D. Johnson
Cell-type memory in a single-cell eukaryote requires the continuous presence of a specific transcription regulator
cell-type memory,transcriptional feedback loop,Candida albicans,degron
Significance Once a eukaryotic cell type is formed, its identity is typically maintained through many rounds of cell division. In principle, such cell-type memory can be produced by many different mechanisms, but it has been difficult to isolate the intrinsic basis of cell memory from other aspects of gene expression. We performed experiments in a fungal species (Candida albicans, a component of the human microbiome) where a particular cell type is stably maintained for thousands of cell divisions. The results cleanly rule out several popular models for cell-type memory and demonstrate that, in this case, a key transcription factor is needed continuously to maintain cell-type memory—even across a single cell division cycle.
Cell-type memory in a single-cell eukaryote requires the continuous presence of a specific transcription regulator

Once a eukaryotic cell type is formed, its identity is typically maintained through many rounds of cell division. In principle, such cell-type memory can be produced by many different mechanisms, but it has been difficult to isolate the intrinsic basis of cell memory from other aspects of gene expression. We performed experiments in a fungal species (Candida albicans, a component of the human microbiome) where a particular cell type is stably maintained for thousands of cell divisions. The results cleanly rule out several popular models for cell-type memory and demonstrate that, in this case, a key transcription factor is needed continuously to maintain cell-type memory—even across a single cell division cycle.

A central principle of developmental biology holds that a cell type, once formed, can maintain its identity through many rounds of cell division. Several mechanisms can, in principle, produce such cell memory including transcriptional feedback loops, directly inherited chromatin structures, directly inherited DNA modifications (such as methylation), and propagation of prions (1). All of these mechanisms have been implicated in cell memory, but because they also influence gene expression, it has often been difficult to isolate the intrinsic basis of cell memory from the many other aspects of gene expression. Given the current state of knowledge, it seems especially important to rigorously investigate cell memory in a variety of different systems, the goal being to understand those mechanisms that are widely applicable. In this paper, we investigate an example of cell memory in the fungal species Candida albicans, a component of the human microbiome and an opportunistic pathogen of humans. Unlike the great majority of fungal species, C. albicans undergoes a process known as white–opaque switching (2), which allows two different cell types, each of which is heritable for thousands of cell divisions, to arise from a single genome. The switching machinery is highly conserved across clinical isolates of C. albicans and extends to Candida dubliniensis (3), which diverged from a common ancestor with C. albicans some 10 to 20 Mya. All of these observations demonstrate that white–opaque switching is under very strong selection and must therefore be of key importance for those Candida species that retain it. White and opaque cells differ in many ways. They can easily be distinguished by the appearance of their colonies on agar plates or by their cell shapes under a light microscope. Opaque cells mate over a million-fold more efficiently than white cells (4), and the two cell types exhibit different interactions with the immune system and express different metabolic pathways (5–7). Over ten percent of the genome is differentially expressed (more than threefold) between the two cell types (8, 9). The central regulator of the opaque cell type is the Wor1 protein. Cells deleted for the WOR1 gene are locked in the white state, and ectopic expression of WOR1 in white cells causes them to switch en masse to the opaque form (10–12). Wor1 is a transcription regulator with an unusual DNA recognition motif (13, 14) which binds directly to its own control region and thereby activates its own transcription; it also binds to and activates many additional opaque-specific genes (15–17). Although Wor1 is the central regulator of white–opaque switching, other transcription regulators contribute to the stability of the opaque state, once formed (12, 15, 16, 18–23). As determined by gene expression and chromatin immunoprecipitation studies, these regulators form a complex network of nested feedback loops (Fig. 1A). We have proposed that the transcriptional network of Fig. 1A, which is largely dormant in white cells but becomes excited in opaque cells, is inherently self-sustaining and responsible for maintaining “memory” of the opaque state across cell divisions (15); however, this idea has not been explicitly tested or refined, nor have competing models been ruled out. In this paper, we document the consequences of rapidly destroying the Wor1 protein in opaque cells to answer the following questions: Is the continual presence of Wor1 required for maintenance of the opaque state? Can the additional regulators of Fig. 1A provide a “backup” and supply transient memory of the opaque state (perhaps for a limited number of generations) in the absence of Wor1? Could an inherited form of chromatin structure preserve the opaque state in the absence of Wor1 and if so, for how many cell divisions? When Wor1 is destroyed, how does the complex, highly connected gene control circuit of Fig. 1A respond?

We adapted an auxin-mediated degradation system (24) for use in C. albicans. The endogenous Wor1 was tagged with degron and green fluorescent protein (GFP) to allow determining in real time the effects of rapidly degrading Wor1 in opaque cells (Methods). As shown in Fig. 1 B and C, Wor1-degron protein levels in opaque cell culture decreased to ~35% after 1.5 h of auxin treatment and were undetectable after 6 h; in contrast, Wor1-degron protein levels in the same strain remained stable in the absence of auxin (Fig. 1B). In the same experiment, we removed aliquots of the culture, rapidly washed the cells to remove the auxin, and plated cells in the absence of auxin to determine whether or not the cells remained committed to the opaque state. Because white and opaque cells produce easily distinguishable colonies on agar plates, we simply counted them. After 6 h of auxin treatment (when Wor1 protein was undetectable), 100% of colonies exhibited the white colony morphology (Fig. 1D), indicating that the opaque state could not be recovered when the auxin was removed at this point. As a control, the parallel culture maintained in the absence of auxin gave rise to 100% opaque colonies (Fig. 1D and SI Appendix, Fig. S1A). At earlier points in the time course of auxin treatment, the percentage of cells that remained in the opaque state (monitored by colony counts after washing, as described above) gradually decreased and correlated well with the Wor1-degron protein levels; there is a consistent delay, on average, of approximately 1 h between the percentage of Wor1 remaining in the culture and the percentage of cells that retain memory of the opaque state (Fig. 1C). This lag is shorter than the generation time for opaque cells in this growth medium (90 min), and we interpret these results as meaning that, without Wor1, the opaque state cannot be reliably transmitted across even a single cell complete division cycle. This interpretation relies on control experiments (SI Appendix, Fig. S1B and Supplementary Text) showing that the auxin-free wash rapidly reverses the protein destruction caused by the degron, consistent with previous reports of rapid auxin reversibility in Saccharomyces cerevisiae (25).

An important insight into the maintenance of the opaque state can be gleaned from the developed colonies (Fig. 1C), especially at the intermediate timepoints (i.e., 1 to 3 h). After washing the cells to remove the auxin, over 99% of the colonies that developed were either purely white or purely opaque across the entire time course. Thus, when Wor1 is partially degraded (on a bulk culture basis), individual cells either maintained or lost memory of the opaque state but did not produce mixed-state colonies. To obtain a higher-resolution view of the effects of degrading Wor1, we used a microfluidic perfusion system coupled with fluorescence microscopy to monitor the Wor1 protein levels in individual cells. The experiment began with opaque cells (showing their characteristic elongated shapes) with strong nuclear GFP signals from the Wor1-degron protein (Fig. 2, “0” timepoint). By 10 h of auxin treatment, the culture was dominated by white cells (easily recognized by their yeast-like budding morphology) with no detectable GFP signal (Fig. 2 and Movie S1). Consistent with the bulk western blot results (Fig. 1B), the level of GFP signal observed in individual cells decreased continuously over time after auxin addition. However, clear heterogeneity in the GFP signal was observed from one cell to the next (e.g., 2-h timepoint in Fig. 2), indicating that, following auxin treatment, individual cells display different levels of Wor1. To test the fate of cells with different levels of Wor1, we used the same microfluidic system as described above, but instead of continuous flow of auxin, we utilized a 2-h pulse of auxin, followed by a wash step in the auxin-free medium. (The continuous presence of auxin and the absence of auxin (Fig. 3 A and C, respectively) were included as controls.) After 2 h of auxin treatment, the fields of cells show heterogeneous levels of GFP signal (Fig. 3B and Movie S2), consistent with previous results (Fig. 2); for example, those cells at the top of field in Fig. 3B are brighter than those at the bottom. Following the shift to auxin-free medium, those cells with brighter GFP signal returned to their original high GFP levels, and all their progeny cells maintained the opaque cellular morphology and high GFP signal. In contrast, cells with lower levels of GFP at 2 h (e.g., those at the bottom of the field) continued to lose the GFP signal after the auxin-free wash; progeny cells eventually lost both fluorescence and opaque cell morphology; that is, they became white cells. This trend—high-expressing cells remained opaque after the auxin-free wash and low-expressing cells did not recover the opaque state following the wash––was observed in many independent microfluidic chambers (SI Appendix, Fig. S2). These results show that, to maintain the opaque state after transient auxin treatment, a critical threshold level of Wor1 is needed. We can make a rough estimate of the threshold concentration of Wor1 needed to maintain the opaque state. As shown in Fig. 1C, when the bulk culture level of Wor1 has decreased to 75% of the original level (after 45 min of auxin treatment), 100% of the cells remain opaque after washing, indicating that, on average, an opaque cell can withstand at least a 25% drop in the Wor1 protein level without irreversibly switching to a white cell. At our next timepoint (1.5 h), the Wor1 protein has dropped to approximately 35% of its opaque level, and there is a significant loss of opaque cell memory. To more directly visualize the threshold levels of Wor1, we tracked 36 individual cells exposed to 4 h of auxin treatment followed by a auxin-free wash step in the microfluidic device (Fig. 4). In this experiment, approximately half of the cells remained opaque and half reverted to white following the wash. We can estimate the threshold needed to maintain the opaque state by comparing the distributions of Wor1 GFP signal at the end of the auxin exposure between cells that retained the opaque state to those that switched to the white state. If we normalize to the range of fluorescence between white cells and opaque cells (as in Fig. 4C), we estimate the threshold as approximately 25% of typical opaque levels. As we cannot differentiate low levels of Wor1 GFP from autofluorescence, this constitutes a lower limit for the threshold. Considering absolute fluorescence values (as shown in the example in Fig. 4A), the threshold is estimated as 70% of typical opaque values, which is the upper limit of the threshold concentration. These values are consistent with those observed in the bulk culture experiments where Wor1 levels were determined directly by western blot rather than fluorescence (Fig. 2). Taking all these observations together, we estimate the threshold level of Wor1 to be between 25% and 75% of its level in normal opaque cells.

The microfluidic system also allowed tracking of the changes in the appearance of individual cells as they switched from opaque to white in response to Wor1 degradation. After the Wor1-degron-GFP protein became undetectable in a mother cell, even the daughter cells (which also lack the GFP signal) did not immediately exhibit the characteristic white cell morphology (yeast-like, budding cells); rather, they displayed an intermediate morphology–shorter than the original opaque cells, but easily distinguishable from true white cells (Fig. 2). Subsequent generations of cells became shorter and shorter and, by three generations, the cells exhibited the conventional white-cell morphology. Thus, upon Wor1 degradation, memory of the opaque state is lost within an hour, but several generations are required for remodeling of the cells to be completed. This lag is not surprising as, among other features, the cell wall structure of white and opaque cells differs dramatically in appearance (2). As described earlier, white and opaque cells of C. albicans have significantly different gene expression profiles, commensurate with their many phenotypic differences. The rapid degradation of Wor1 allowed us to order the gene expression changes that occur when cells switch from opaque to white. We performed RNAseq in Wor1-degron opaque cells at 8 timepoints across 6 h of auxin treatment (SI Appendix, Figs. S1C, S3, and S4 and Supplementary Text). Obviously, there is a great deal of information from these experiments, and here we mention only a few observations. First, levels of Wor1 mRNA rapidly decline upon auxin treatment, consistent with previous work showing that the Wor1 protein directly activates its own transcription (17). Second, the other activators of white–opaque switching (Fig. 1A) show different, idiosyncratic patterns; for example, WOR3 mRNA declines even faster than WOR1 mRNA, CZF1 declines slowly, and WOR4 transiently increases before slowly declining. Fourth, the bulk of the genes that change are white- or opaque-enriched genes and can easily be grouped into early, middle, and late clusters depending on their responses to Wor1 destruction (SI Appendix, Supplementary Text). Finally, by 6 h of auxin treatment, the transcription pattern closely resembled that of true white cells, but some opaque-specific mRNAs had not fully declined to their white cell levels (SI Appendix, Fig. S3), consistent with the lag in cell morphology observed in the microfluidic experiments.

In this paper, we address how a particular cell type (the opaque cell) of the fungal species C. albicans is faithfully maintained across thousands of cell generations. The formation and maintenance of this cell type is “epigenetic” in the sense that the genome sequence remains the same as that of the other cell type, the white cell (26). Our strategy relies on rapidly degrading the key transcription regulator of the opaque state (Wor1) and monitoring the consequences using fluorescence microscopy, RNA-seq, cell-type commitment plating assays, and other approaches. The principal conclusions of this paper are as follows: 1. Within an hour of auxin-mediated Wor1 degradation, opaque cells irreversibly switch to the white cell state; that is, they cannot be rescued after this point by removing the auxin and halting the destruction. In these experiments, the doubling time of cells is 90 min; therefore, memory of the opaque state cannot be transmitted across even a single complete round of cell division, much less the thousands of generations typical of the opaque state inheritance. 2. A threshold level of Wor1, estimated to be between 25% and 75% of the normal level of Wor1 in opaque cells, is needed to maintain the opaque state. Although there is some degree of robustness of the opaque state to fluctuating levels of Wor1, this tolerance is clearly limited in degree. 3. Following Wor1 degradation and the irreversible loss of opaque cell memory, several additional generations are required for the transitioning opaque cells to fully assume the morphological appearance and mRNA expression pattern of true white cells. 4. When Wor1 protein is degraded, its mRNA rapidly declines, but the mRNAs of the additional transcription regulators of the opaque cell circuit (Fig. 1A) do not decline in concert with Wor1. Some of these mRNAs retain their opaque cell levels well after Wor1 has been degraded (and some even transiently increase), but this sustained synthesis is not sufficient to provide even a temporary backup mechanism to maintain memory of the opaque state. These results demonstrate that there can be no other mechanism for cell memory that functions independently of the Wor1 protein. As far as is known, Wor1 does not have any specialized properties other than being a sequence-specific DNA binding protein that regulates transcription. It follows that models such as directly inherited chromatin structures or directly inherited DNA modifications cannot independently provide opaque cell memory across even a single complete cell division cycle. These mechanisms may well come into play to fully express the opaque state, but, on their own, they cannot explain how the memory of this state is maintained. We note that Wor1 is believed to form a biomolecular condensate with most of the other transcription regulators of Fig. 1A (27); our results show that this proposed condensate does not protect Wor1 from auxin-mediated degradation, nor can any remaining condensate be a source of cell memory independent of Wor1. A model that is consistent with all the observations in this paper is a relatively simple transcription feedback model for cell memory, where Wor1 protein activates its own synthesis and is transmitted to daughter cells, sustaining the feedback loop across cell divisions. There is a well-studied precedent for a single transcriptional feedback loop providing stable cell memory across thousands of generations, namely the bacteriophage lambda repressor, which holds an integrated prophage in check while activating its own synthesis (28). A conceptionally similar, artificial transcriptional feedback loop in S. cerevisiae has been described that also exhibits cell memory (29, 30), indicating that there is no theoretical barrier to this solution in eukaryotes. Our work shows that a similar principle can explain a much more complex, naturally occurring system, one that underlies a large cell-type program in a fungal pathogen. The C. albicans experiments described here provide several advantages over other studies of cell memory. For example, removal of a key cell type–specific transcription regulator from animal cells has been typically carried out by blocking (sometimes only partially) new synthesis of the protein, often requiring relatively long times for the protein itself to decline. An exception is Bates et al. where Oct4 was rapidly degraded in pluripotent mouse stem cells using a similar auxin-mediated system (31). Rapid depletion of Oct4 was shown to affect the expression of many genes involved in pluripotency, but the point at which the stem cells irreversibly lost pluripotency was not reported. Serrano-Saiz et al. showed that the transcription regulators unc-86 (in worms) and Brn3a (in mice) are continually required to maintain the “neuron-class” differentiated cell type (32). And the C. elegans transcription regulator che-1 was shown to require a positive feedback loop to maintain a particular type of neuron (33). An important difference between these experiments and ours is that maintenance of the differentiated state in these postmitotic cells does not require that the memory persists across cell divisions. Our experiments directly address a case where cell memory remains stable across many cycles of cell division. Given that this basic mechanism—a self-sustaining positive feedback loop—is found across diverse settings in bacteria, fungi, and animals, it seems likely that this same basic mechanism will be applicable to many other cases of cell memory.

The WOR1-degron opaque cells were grown in SD+aa+uri until the cell density reached 0.20 OD/mL. Auxin (3-Indoleacetic acid, Sigma-Aldrich) was then added at a final concentration of 100 µM. After treatment, an aliquot of the culture was taken from the culture and analyzed for Wor1 (by western blots). Another aliquot of the culture was washed and plated on the SD+aa+uri agar plate, followed by the counting of white and opaque colonies to determine the population of these two cell types. To monitor the changes at the single-cell level in real time, a microfluidic perfusion system coupled with fluorescence microscopy was used.

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