In this paper, we have dissected the genetic circuitry controlling white-opaque switching in the fungal pathogen C. albicans. White-opaque switching is an epigenetic change between two distinct types of cells, both containing the same genome. The white-opaque switch is crucial for many aspects of C. albicans biology, including interactions with other C. albicans cells (pheromone sensing and mating) and interactions with the host (opportunistic pathogenesis). Our results are summarized in and , where the circuitry controlling this switch is diagrammed. This network of positive feedback loops is responsible for the heritability of each state, as well as the frequency of switching between them, and we propose that the structure of this network makes an important contribution to the biology of white-opaque switching.
Model of the Genetic Network Regulating the White-Opaque Switch
Activity of the White-Opaque Genetic Regulatory Network in Different Cell Types
The default state can be considered the white cell type: most clinical isolates of C. albicans
/α cells, and they are locked in the white state through a1-α2 repression of WOR1
(A). However, even in a
and α cells, which are permissive for white-opaque switching, the white cell type still seems to be the default, in that white cells are generally more stable than opaque cells (B). For example, opaque cells at 24 °C are stable for many generations, but above 30 °C they become unstable and rapidly switch back en masse to the white form, which is stable under these conditions [1
]. There are no known environmental conditions that comparably destabilize the white form. In our model, the opaque form is generated when the series of positive feedback loops shown in become excited (C). Thus, in opaque cells, WOR1
likely directly induces CZF1
expression, and in turn, CZF1
both activate WOR1
does this by repressing a repressor of the opaque state (EFG1
), the net effect being a positive feedback loop.
The multiple feedback loops observed in the opaque state are reminiscent of those seen in differentiated animal cells, such as those of the Drosophila
], reviewed in [23
]) and the mammalian myoblast ([24
], reviewed in [25
]). A series of such feedback loops (as opposed to a single loop) buffers the circuit against transient fluctuations in any single regulatory protein and therefore provides additional stability to the excited form of the circuit. In addition, the nature of the circuit probably defines the switching frequency. For example, deletion of CZF1
decreases the white-to-opaque switching frequency by approximately 10-fold, but has little effect on the backward switching rate. Thus, the primary role of CZF1
seems to be in modulating the switching frequency; in contrast, WOR1
are both required to maintain the opaque state; thus their roles are more integral to the switch itself.
Although the overall logic of the circuit shown in can explain many features of white-opaque switching, there appear to be several unusual features of the circuit components themselves that likely also play important roles in white-opaque switching. For example, our ChIP-chip experiments revealed that Wor1 binding shows a bias toward genes with unusually long upstream intergenic regions—as defined by the distance from the 5′ end of the ORF to the next annotated coding region. This observation suggests that these genes bound by Wor1, which include the four encoding transcriptional regulators that form the interlocking feedback loops (WOR1
, and WOR2
) are also controlled by a number of other transcriptional regulators. It is known that the frequency of white-opaque switching can be influenced by environmental cues (e.g., temperature and oxidative stress) [1
], and it seems plausible that different rates of switching could be “set” by individually adjusting the levels of the regulatory proteins that make up the circuit. For example, since deletion of CZF1
reduces the frequency of white-to-opaque switching 10-fold, regulation of the level of Czf1 by environmental signals could directly control the “forward” switching rate. Another unusual feature of the circuit concerns the wide distribution of Wor1 over much of the upstream regions of WOR1, EFG1, CZF1,
(), suggesting a highly cooperative transcriptional response to the intracellular levels of WOR1
. This, combined with the interlocking positive feedback loops, could be responsible for the switch-like behavior of the system, specifically the failure to readily observe a cell type intermediate between white and opaque in wild-type switching strains.
The switch from the white to the opaque form growth alters transcription of approximately 400 genes. We know that the master regulator Wor1 ultimately controls all of these genes, since deletion of WOR1
locks cells in the white form, and ectopic expression of WOR1
converts white cells en masse to opaque cells. Our ChIP-chip analysis revealed that Wor1 directly regulates approximately 15% of this gene set (20 white-enriched genes and 38 opaque-enriched genes). Since Wor1 is also bound upstream of CZF1, EFG1, WOR2,
and 20 additional transcriptional regulators (see Table S1
), it seems likely that much of white-opaque switching program is regulated indirectly by Wor1 through its effects on other transcriptional regulators.
An unexpected outcome of the Wor1 ChIP-chip experiments was the presence of Wor1 at a large number of genes that were not identified as white- or opaque-enriched in previous microarray analyses [3
]. There are several explanations for this observation. First, Wor1 could control these genes in both white and opaque cells, with their transcription being unaffected by the white-to-opaque transition. We think this explanation is unlikely because Wor1 is up-regulated 45-fold in opaque cells [3
], and it seems improbable that this change could have no impact on expression of target genes. To test this idea directly, we performed a Wor1 ChIP-chip experiment in white a
cells, and found that Wor1 is not bound at any of these target genes (unpublished data). A second possibility, one that we favor, is that Wor1 may occupy the promoters of these 100 genes in opaque cells, preparing their expression to respond to unknown environmental signals, perhaps those generated by the host. According to this idea, the standard laboratory conditions used for transcriptional profiling would not have included the necessary environmental stimuli, and thus these genes would not have been identified as regulated by the white-opaque switch. This idea suggests there are additional aspects to white-opaque switching which have not been previously recognized.
Finally we note that white-opaque switching does not appear to be a general feature of fungi, even those that are closely related to C. albicans
. Indeed it may have arisen during the long association of C. albicans
with its warm-blooded hosts. The evolution of a complex circuit composed of interlocking feedback loops is relatively simple to imagine, as it could occur stepwise simply through the acquisitions of cis-acting sequences in genes for transcriptional regulators used for other purposes in the cell. We note that Czf1 also relieves Efg1-mediated repression of hyphal growth under embedded conditions [27
], and this genetic relationship has been maintained in the regulation of the white-opaque switch. Thus EFG1
have other key functions in the cell—even in cells that are genetically blocked for white-opaque switching—and their involvement in white-opaque switching could well be a recent adaptation, functioning to modulate the stability of the two states and the frequency of switching between them. The independent evolution of interlocking transcriptional feedback loops in a variety of distinct biological contexts (white-opaque switching in C. albicans,
eye development in flies, and muscle development in mammals, for example) suggests they are particularly effective ways of providing, from the same genome, distinctive cell types that can be stably propagated for many generations.