Completing the Candida Loop

2100 Imagine identical twin sisters, one short, dark-haired, and zaftig; the other tall, blond, and willowy. You’re not likely to run across this pair, since identical twins possess matching genomes and thus, when raised under the same conditions, look alike. But some organisms, like the fungus Candida albicans, can assume dramatically different, heritable forms even though they share identical genes. A common human pathogen, C. albicans , most commonly exists in what is known as the white form—rounded yeast cells that grow as hemispheric white colonies. But in rare cases, it spontaneously switches to a less-stable opaque form, with elongated cells and fl at, grayish colonies. The switch to the opaque form not only changes the fungus’s appearance, but more importantly permits C. albicans cells to interact differently, both with each other and with their mammalian hosts. White cells fare better when infecting the bloodstream, for example, while opaque cells are more optimized for colonizing skin. Previous research has shown that two genes, white-opaque regulator 1 ( WOR1 ) and enhanced fi lamentous growth 1 ( EFG1 ), are involved in white-opaque switching. In a new study, Rebecca Zordan, Mathew Miller, and colleagues fl eshed out the circuitry responsible for the C. albicans whiteopaque switch, identifying two genes that, along with EFG1 and WOR1 , make up a network of positive-feedback loops. To do this, they started with over 400 genes that are expressed differently in white and opaque cells. They eventually focused on two genes, one that they named WOR2 and one called CZF1 , which had been previously studied in other processes in C. albicans . Both of these genes show increased expression in opaque cells and, like WOR1 and EFG1 , code for transcription factors (proteins that, by binding to DNA, regulate when, where, and how much RNA is synthesized from specifi c genes). To determine whether WOR2 and CZF1 are involved in regulating the white-opaque switch, the authors created C. albicans mutants in which either WOR2 or CZF1 was deleted. Deletion of either of these genes dramatically reduced the rate of whiteopaque switching, indicating that, in nonmutant cells, they function as switch activators. When an extra dose of Czf1 was introduced into nonmutant white cells (a technique known as ectopic expression), nearly all of the cells switched to the opaque form. This was not the case when WOR2 was ectopically expressed, indicating that additional Wor2 is not suffi cient to activate switching. Next, the authors wanted to fi nd out how CZF1 and WOR2 interact with WOR1 and EFG1 , the previously identifi ed genes, to regulate switching. Using a series of mutants with either one or two genes deleted (along with ectopic expression), the authors deduced that WOR1 regulated both WOR2 and CZF1 , since ectopic expression of WOR1 caused increased white-opaque switching even when WOR2 or CZF1 were deleted. It was known that EFG1 is required to stably maintain the white state from one generation to the next. The authors now propose that EFG1 is repressed by CZF1 in opaque cells. To test whether the WOR1 protein directly interacts with the DNA of the other three genes in opaque cells, the authors performed a series of experiments (called chromatin immunoprecipitation assays) to determine whether a given protein binds to or is localized to a specifi c DNA sequence. They found that—in addition to binding to the gene regulatory regions of CZF1 , WOR2 , and EFG1 — WOR1 binds to the regulatory region of its own gene and to the regulatory regions of about 60 other genes that are differentially expressed in white and opaque cells, providing more evidence that WOR1 is a “master regulator” of switching. The authors present a model of the circuit controlling the white-opaque switch in which WOR1 directly induces CZF1 and WOR2 expression, which in turn activates WOR1 expression. ( CZF1 does this in a roundabout manner by repressing EFG1 , which serves as a repressor of white-opaque switching.) The net result is a complex series of positive-feedback loops that give rise to two heritable states. When the feedback loops are inactive, C. albicans assumes the white form; excitation of the loops drive C. albicans into the opaque state. The regulators of this loop are produced at relatively high levels and are presumably inherited by daughter cells, thus ensuring that the loop remains active in progeny cells. A series of interlocking loops (as opposed to a single loop) may buffer the switch against minor fl uctuations in the levels of any one component, thereby providing some additional stability to opaque cells as they divide. The authors observe that this gene-regulatory circuit is similar in principle to the transcriptional feedback loops seen in certain animal developmental processes. That something so complex evolved independently to regulate processes as disparate as eye development in fl ies, muscle development in mammals, and white-opaque switching in C. albicans suggests that the circuit is an effi cient way of using one genome to endow cells with very different properties and of ensuring that these new properties will be stably inherited from generation to generation. The C. albicans whiteopaque switch is undoubtedly just one example among many similar circuits that have a large and inherited effect on cells that remain to be discovered.