The human pathogen Candida albicans can assume either of two distinct cell types, designated “white” and “opaque.” Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions.

A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Here, we report the identification of two adaptation-defective mutants that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This adaptation-defective phenotype was entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest. We show that one mutation resides in CDC5, which encodes a polo-like kinase, whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II.

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.phenotypic switching ͉ transcriptional regulation ͉ WOR1 I n this article, we examine the interconversion between two distinctive types of cells in the human fungal pathogen Candida albicans. This interconversion, which plays important roles in both pathogenesis and mating, exemplifies two characteristics shared by many examples of cell differentiation: The conversion from one cellular state to another is stochastic, and each state, once formed, is heritable for many generations.The property of C. albicans we investigate is called whiteopaque switching, and it refers to an alternation between two distinctive types of cells, white and opaque (1). White cells generally give rise to white cell progeny, but, approximately every 10,000 generations, a white cell spontaneously switches to the opaque form, which then will produce opaque cell progeny for many generations (2, 3). Conversely, an opaque cell can spontaneously switch back to the white form, and the progeny of this cell will remain in the white form for many generations. Any molecular mechanism for white-opaque switching therefore must account for the ability of each state to stochastically convert to the other as well as the heritability of each state, once formed.White and opaque cells of C. albicans differ in many features (for reviews, see refs. 4-6). They are easily distinguished under the microscope, with white cells appearing nearly spherical and opaque cells appearing larger and more elongated. When grown on agar plates, white cells form white, dome-shaped colonies, whereas opaque colonies are darker and lie flatter against the agar. Many, if not all, of the differences between white and opaque cells are due to differences in gene expression: For example...

It is widely suspected that gene regulatory networks are highly plastic. The rapid turnover of transcription factor binding sites has been predicted on theoretical grounds and has been experimentally demonstrated in closely related species. We combined experimental approaches with comparative genomics to focus on the role of combinatorial control in the evolution of a large transcriptional circuit in the fungal lineage. Our study centers on Mcm1, a transcriptional regulator that, in combination with five cofactors, binds roughly 4% of the genes in Saccharomyces cerevisiae and regulates processes ranging from the cell-cycle to mating. In Kluyveromyces lactis and Candida albicans, two other hemiascomycetes, we find that the Mcm1 combinatorial circuits are substantially different. This massive rewiring of the Mcm1 circuitry has involved both substantial gain and loss of targets in ancient combinatorial circuits as well as the formation of new combinatorial interactions. We have dissected the gains and losses on the global level into subsets of functionally and temporally related changes. One particularly dramatic change is the acquisition of Mcm1 binding sites in close proximity to Rap1 binding sites at 70 ribosomal protein genes in the K. lactis lineage. Another intriguing and very recent gain occurs in the C. albicans lineage, where Mcm1 is found to bind in combination with the regulator Wor1 at many genes that function in processes associated with adaptation to the human host, including the white-opaque epigenetic switch. The large turnover of Mcm1 binding sites and the evolution of new Mcm1–cofactor interactions illuminate in sharp detail the rapid evolution of combinatorial transcription networks.

The budding yeast Saccharomyces cerevisiae has three cell types (a cells, ␣ cells, and a͞␣ cells), each of which is specified by a unique combination of transcriptional regulators. This transcriptional circuit has served as an important model for understanding basic features of the combinatorial control of transcription and the specification of cell type. Here, using genome-wide chromatin immunoprecipitation, transcriptional profiling, and phylogenetic comparisons, we describe the complete cell-type-specification circuit for S. cerevisiae. We believe this work represents a complete description of cell-type specification in a eukaryote.chromatin immunoprecipitation ͉ mating ͉ transcriptional circuit A problem of central importance in understanding multicellular organisms is how different cell types are stably maintained. Typically, cell-type specification is based on a transcriptional circuit in which combinations of regulatory proteins determine the final pattern of gene expression that is appropriate to a given cell type. Although unicellular, the yeast Saccharomyces cerevisiae has three distinct types of cells, and the cell-specification circuit is combinatorial (refs. 1-3 and Fig. 1). The a and ␣ cell types are typically haploid in DNA content and mate with each other in an elaborate ritual that culminates in cellular and nuclear fusion. These events produce the third type of cell, the a͞␣ cell type, which is typically diploid. This cell type cannot mate but, when environmental conditions are appropriate, can undergo meiosis and sporulation, producing two a and two ␣ cell types. The patterns of cell-type-specific gene expression are set up by a few sequence-specific DNA-binding proteins acting in various combinations. Three critical proteins (␣1, ␣2, and a1) are encoded by the mating-type (MAT) locus. A fourth key sequence-specific DNA-binding protein (Mcm1) is encoded elsewhere in the genome. In this article we use the term ''cell-type-specification circuit'' to refer to the regulatory scheme diagrammed in Fig. 1, because each component and branch of this scheme is necessary and sufficient to establish and maintain three cell types.In this article we apply three methods [genome-wide chromatin immunoprecipitation (ChIP), genome-wide transcriptional profiling, and phylogenetic comparisons] in an attempt to completely determine the cell-type-specification circuit in S. cerevisiae (4-8). The use of three different techniques generated considerably more data than are needed to reconstruct the circuit, and because it is overdetermined, we believe our circuit description to be very accurate, containing at most only a few false negatives or positives. Figs. 3, 5 A and B, and 6. EG123, yDG208, and yDG240 are all derivatives of S288C. For the salt-sensitivity experiment, the a1-␣2 site in the endogenous HOG1 gene promoter was replaced by integration of Kluyveromyces lactis URA3, which was subsequently replaced by the integration of an oligonucleotidegenerated construct to restore the HOG1 promoter with a modified a1-...