A 368-Base-Pair cis -Acting HWP1 Promoter Region, HCR, of Candida albicans Confers Hypha-Specific Gene Regulation and Binds Architectural Transcription Factors Nhp6 and Gcf1p

Abstract

To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation.Candida albicans is an opportunistic fungal pathogen that rapidly transitions between yeast, true hyphal, and pseudohyphal growth forms in response to a number of environmental stimuli, including temperature, pH, and medium composition (15,19,21). Changes in the external environment of C. albicans are known to be accompanied by changes in the genetic expression patterns of morphology-specific transcripts, leading in turn to the activation and deactivation of genetic pathways that are believed to underlie the multiple interactions of C. albicans with its mammalian hosts, including the colonization and invasion of tissue and resistance to host defenses (16,28).The true hyphal morphology of C. albicans (43) is notable for its enhanced adherence in comparison to yeast forms (40), and this is reflected in the presence of hypha-specific proteins that promote the invasion of and adherence to host surfaces. One such protein, Hwp1 (hyphal wall protein 1), is a glycosylphosphatidylinositol-linked cell wall protein that is abundantly expressed when C. albicans is in hyphal growth mode and is absent or barely detectable in the yeast morphology (41). Hwp1 is incorporated into the cell wall at the tips of apically growing hyphae, limiting its presence to hyphal surfaces (39).Hwp1 has been shown to play a key role in the attachment of C. albicans to human buccal epithelial cells by forming covalent bonds through its function as a substrate for epithelial transglutaminases (40). HWP1 gene expression is regulated at the level of mRNA. Northern blot analysis reveals abundant steady-state levels of HWP1 mRNA during hyphal growt...

Efg1-mediated Recruitment of NuA4 to Promoters Is Required for Hypha-specific Swi/Snf Binding and Activation inCandida albicans

“…Among them, surrounding regions were shown in the Figure 3A. Importantly, the Efg1-bound regions on the HWP1 and ALS3 promoters coincide exactly with the recently mapped regions essential for the induction of HWP1 and ALS3 (Argimon et al, 2007;Kim et al, 2007). Interestingly, Efg1 was detected at the promoters in both yeast and hyphal states ( Figure 3A).…”

“…Although the UAS regions for HWP1 and ALS3 have been mapped (Argimon et al, 2007;Kim et al, 2007), how Efg1 regulates their expression has not been elucidated. In this study, we identify an orthologue of the NuA4 subunit as an Efg1 interacting protein in a two-hybrid screen.…”

“…ECE1, HWP1, and ALS3 are the first few hypha-specific genes identified (Birse et al, 1993;Hoyer et al, 1998;Staab and Sundstrom, 1998;Zhao et al, 2004). Although the UAS regions for HWP1 and ALS3 have been mapped (Argimon et al, 2007;Kim et al, 2007), how Efg1 regulates their expression has not been elucidated. In this study, we identify an orthologue of the NuA4 subunit as an Efg1 interacting protein in a two-hybrid screen.…”

Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans

“…The nuclear isoform may participate in gene regulation and the mitochondrial isoform in packaging of mtDNA and/or mtDNA metabolism. However, because Kim et al (2007) used crude protein extracts as a source for purification of proteins binding to the promoter region, the binding of Gcf1p to the nuclear sequence might have been artefactual. For example, Gcf1p as a non-specific DNA-binding protein could have been released from mitochondria upon preparation of protein extracts, resulting in its binding to the Hwp1 promoter in vitro.…”

The Yak1 Kinase Is Involved in the Initiation and Maintenance of Hyphal Growth inCandida albicans

“…These negative regulators of filamentation act independently of Efg1 and other positive regulators of filamentation (Braun and Johnson, 1997;Braun et al, 2001;Kadosh and Johnson, 2001;Murad et al, 2001b). The interplay between positive and negative regulation of the yeast-to-hypha switch is illusThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09 -0960) on March 5, 2008. trated by the complex structure of the promoter regions of hypha-specific genes such as ALS3 and HWP1, which integrate inputs from multiple activators and repressors and are under the control of chromatin remodeling proteins (Argimon et al, 2007;Kim et al, 2007). To date, the exact relationships, interconnections, and feedback regulations of the signaling pathways and transcription factors that positively and negatively regulate filamentation remain largely uncharacterized.…”