Sin3 and Rpd3 negatively regulate a diverse set of yeast genes. A mouse Sin3-related protein is a transcriptional corepressor, and a human Rpd3 homolog is a histone deacetylase. Here, we show that Sin3 and Rpd3 are specifically required for transcriptional repression by Ume6, a DNA-binding protein that regulates genes involved in meiosis. A short region of Ume6 is sufficient to repress transcription, and this repression domain mediates a two-hybrid and physical interaction with Sin3. Coimmunoprecipitation and two-hybrid experiments indicate that Sin3 and Rpd3 are associated in a complex distinct from TFIID and Pol II holoenzyme. Rpd3 is specifically required for repression by Sin3, and artificial recruitment of Rpd3 results in repression. These results suggest that repression by Ume6 involves recruitment of a Sin3-Rpd3 complex and targeted histone deacetylation.

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of “virulence factors” important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

contributed equally to this workIn response to a variety of external signals, the fungal pathogen Candida albicans undergoes a transition between ellipsoidal single cells (blastospores) and ®la-ments composed of elongated cells attached end-toend. Here we identify a DNA-binding protein, Nrg1, that represses ®lamentous growth in Candida probably by acting through the co-repressor Tup1. nrg1 mutant cells are predominantly ®lamentous under non-®lament-inducing conditions and their colony morphology resembles that of tup1 mutants. We also identify two ®lament-speci®c genes, ECE1 and HWP1, whose transcription is repressed by Nrg1 under noninducing conditions. These genes constitute a subset of those under Tup1 control, providing further evidence that Nrg1 acts by recruiting Tup1 to target genes. We show that growth in serum at 37°C, a potent inducer of ®lamentous growth, causes a reduction of NRG1 mRNA, suggesting that ®lamentous growth is induced by the down-regulation of NRG1. Consistent with this idea, expression of NRG1 from a non-regulated promoter partially blocks the induction of ®lamentous growth.

Candida albicans, the major human fungal pathogen, undergoes a reversible morphological transition from blastospores (round budding cells) to filaments (elongated cells attached end-to-end). This transition, which is induced upon exposure of C. albicans cells to a number of host conditions, including serum and body temperature (37°C), is required for virulence. Using whole-genome DNA microarray analysis, we describe 61 genes that are significantly induced (>2-fold) during the blastospore to filament transition that takes place in response to exposure to serum and 37°C. We next show that approximately half of these genes are transcriptionally repressed in the blastospore state by three transcriptional repressors, Rfg1, Nrg1, and Tup1. We conclude that the relief of this transcriptional repression plays a key role in bringing the C. albicans filamentous growth program into play, and we describe the framework of this transcriptional circuit. INTRODUCTIONThe yeast Candida albicans is the major human fungal pathogen. Although normally present as a commensal organism in the human digestive tract, Candida can cause oral and vaginal thrush, as well as a variety of more serious mucosal and systemic infections. Unlike most pathogens, Candida is capable of infecting all the major organs and tissues of the human body and has no known reservoir outside of warmblooded animals (Odds, 1988(Odds, , 1994a(Odds, , 1994bDupont, 1995;Weig et al., 1998). Candidiasis is now the fourth-leading cause of hospital-acquired infections in the world with an attributable mortality of up to 35% (Edmond et al., 1999). Immunocompromised individuals, such as cancer patients undergoing chemotherapy, AIDS patients, and organ transplant recipients are particularly susceptible to Candida infections (for reviews see Shepherd et al., 1985;Dupont, 1995;Weig et al., 1998). Currently, approximately $1 billion per year is spent on the treatment of patients with hospitalacquired infections in the United States alone (Miller et al., 2001).Several properties of C. albicans are known to contribute to its virulence. These include adhesiveness to host cells, secretion of degradative enzymes, and-the subject of this article-the ability to undergo a reversible morphological transition from the blastospore (single-celled) to filamentous (elongated cells attached end-to-end) forms (Lo et al., 1997;Mitchell, 1998;Brown and Gow, 1999;Ernst, 2000aErnst, , 2000bCalderone and Fonzi, 2001;Brown, 2002b;Calderone and Gow, 2002;Saville et al., 2003). The filamentous forms of C. albicans encompass two distinct morphologies, pseudohyphae and hyphae. In pseudohyphae, the cells are attached end-to-end but each cell has an elliptical shape with constrictions at the septa. In true hyphae, these constrictions are absent and a row of cells show a relatively uniform width. Pseudohyphae and hyphae also differ in other respects, including the precise ways that mitosis and cell division are carried out (Odds, 1985(Odds, , 1988Sudbery et al., 2004). In this article, we use the term filam...

The specific ability of the major human fungal pathogen Candida albicans, as well as many other pathogenic fungi, to extend initial short filaments (germ tubes) into elongated hyphal filaments is important for a variety of virulence-related processes. However, the molecular mechanisms that control hyphal extension have remained poorly understood for many years. We report the identification of a novel C. albicans transcriptional regulator, UME6, which is induced in response to multiple host environmental cues and is specifically important for hyphal extension. Although capable of forming germ tubes, the ume6⌬/ume6⌬ mutant exhibits a clear defect in hyphal extension both in vitro and during infection in vivo and is attenuated for virulence in a mouse model of systemic candidiasis. We also show that UME6 is an important downstream component of both the RFG1-TUP1 and NRG1-TUP1 filamentous growth regulatory pathways, and we provide evidence to suggest that Nrg1 and Ume6 function together by a negative feedback loop to control the level and duration of filament-specific gene expression in response to inducing conditions. Our results suggest that hyphal extension is controlled by a specific transcriptional regulatory mechanism and is correlated with the maintenance of high-level expression of genes in the C. albicans filamentous growth program.