Transcriptional loops meet chromatin: a dual-layer network controls white-opaque switching in Candida albicans - PubMed (original) (raw)

Transcriptional loops meet chromatin: a dual-layer network controls white-opaque switching in Candida albicans

Denes Hnisz et al. Mol Microbiol. 2009 Oct.

Free PMC article

Abstract

The human pathogen Candida albicans is able to undergo a reversible switch between two distinct cell types called white and opaque, which are considered different transcriptional states of cells harbouring identical genomes. The present model of switching regulation includes the bistable expression of a master switch gene that is controlled by multiple transcriptional feedback loops. Here, we show that chromatin-modifying enzymes constitute an additional important regulatory layer of morphogenetic switching. We identify eight chromatin modifiers as switching modulators. Extensive epistasis analysis maps them into at least two independent signalling pathways overlaying the known transcriptional network. Interestingly, we identify the conserved Set3/Hos2 histone deacetylase complex as a key regulator relying on the methylation status of histone H3 lysine 4 for switching modulation. Furthermore, we demonstrate that opaque to white switching is facilitated by the presence of adenine in vitro, but adenine has no effect on switching once the Set3/Hos2 complex is disrupted. Our observations postulate that chromatin modifications may serve as a means to integrate environmental or host stimuli through the underlying transcriptional circuits to determine cell fate in C. albicans.

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Figures

Fig. 1

Fig. 1

WOR1 acts downstream of EFG1 in phase commitment, while EFG1 acts downstream of WOR1 in morphology determination. A. Colony and cellular morphologies on modified Lee's medium containing 5 μg ml−1 Phloxin B. Scale bars correspond to 5 μm (upper panel) and 2 mm (lower panel). B. qRT-PCR analysis of phase-specific mRNA transcripts. WH11, EFG1 (white-specific) and OP4, SAP1 (opaque-specific) transcript levels were normalized to the transcript level of PAT1 (Zordan et al., 2006). qRT-PCR reactions were performed in triplicates and cDNA isolated from two independent cultures were analysed. Data are shown as mean ± SD. C. Immunoblot analysis confirms that WOR1 is repressed by EFG1 and the a/α repressor. Tubulin indicates equivalent loading. D. WOR1 is required for mating. Quantitative mating assays were performed with an opaque phase _MTL_α/α tester strain. At least two independent experiments per genotype were performed yielding qualitatively similar results. Values are shown of one representative experiment. #: tested with both an MTLa/a and an _MTL_α/α tester strain.

Fig. 2

Fig. 2

Histone modifiers act upstream of WOR1. A. Functional categories of single gene deletions on white–opaque switching. B. Immunoblot analysis demonstrates that Wor1 is expressed in a similar pattern in wild type and mutant white (W) and opaque (O) cultures. Tubulin indicates equivalent loading. C. Mating competence is differentially regulated in single mutant cells similar to wild type. Quantitative mating assays were performed with an opaque phase _MTL_α/α tester strain. At least two independent experiments per genotype were performed giving qualitatively similar results. Values are shown of one representative experiment. D. Transcript levels of histone modifiers are phase-independent. qRT-PCR was performed in triplicates and cDNA isolated from two independent cultures were analysed. Transcript levels are normalized to PAT1. Data are shown as mean ± SD.

Fig. 3

Fig. 3

The Set3/Hos2 complex is a key regulator of white–opaque switching. A. The PHD finger of CaSET3. The amino acid sequence was aligned to the PHD fingers of ScSet3, ScPho23, ScYng1, ScCti6 and MmIng2 that were shown to bind H3K4me3 specifically in vitro (Shi et al., 2006; 2007). Colours indicate homologous residues. Arrowheads highlight the residues of the characteristic Cys4-His-Cys3 Zn2+ co-ordination motif. B. qRT-PCR analysis of WOR1 and WOR2 expression in Set3/Hos2-pathway mutants. Deletion of either SET3 or HOS2 in an _efg1_Δ/Δ background does not cause significant changes in the steady-state transcription level of either WOR1 or WOR2. The mRNA levels are normalized to PAT1. qRT-PCR reactions were performed in triplicates and cDNA isolated from two independent cultures were analysed. Data are shown as mean ± SD. C. Dual-layer model of the regulation of white–opaque switching in C. albicans. The dotted grey circle denotes the transcriptional circuit as described (Zordan et al., 2007). White and opaque enriched regulators are shown in white and grey respectively. Coloured elements represent histone-modifying pathways modulating the output of the transcriptional circuit.

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