Global roles of Ssn6 in Tup1- and Nrg1-dependent gene regulation in the fungal pathogen, Candida albicans

Abstract

In budding yeast, Tup1 and Ssn6/Cyc8 form a corepressor that regulates a large number of genes. This Tup1-Ssn6 corepressor appears to be conserved from yeast to man. In the pathogenic fungus Candida albicans, Tup1 regulates cellular morphogenesis, phenotypic switching, and metabolism, but the role of Ssn6 remains unclear. We show that there are clear differences in the morphological and invasive phenotypes of C. albicans ssn6 and tup1 mutants. Unlike Tup1, Ssn6 depletion promoted morphological events reminiscent of phenotypic switching rather than filamentous growth. Transcript profiling revealed minimal overlap between the Ssn6 and Tup1 regulons. Hypha-specific genes, which are repressed by Tup1 and Nrg1, were not derepressed in ssn6 cells under the conditions studied. In contrast, the phase specific gene WH11 was derepressed in ssn6 cells, but not in tup1 or nrg1 cells. Hence Ssn6 and Tup1 play distinct roles in C. albicans. Nevertheless, both Ssn6 and Tup1 were required for the Nrg1-mediated repression of an artificial NRE promoter, and lexA-Nrg1 mediated repression in the C. albicans one-hybrid system. These observations are explained in models that are generally consistent with the Tup1-Ssn6 paradigm in budding yeast.

Figure 1.

Ssn6-like proteins in eukaryotes. (A) S. cerevisiae paradigm of Ssn6-Tup1 function. (B) Arrangement of TPR repeats in Ssn6-like proteins from C. albicans, S. cerevisiae, Ustilago maydis, Schizosaccharomyces pombe, Dictyostelium discoideum, humans, Drosophila melanogaster, and Caenorhabditis elegans. Coordinates of TPR domains are shown, and percentage amino acid sequence similarities between the shaded regions of CaSsn6 and other Ssn6-like proteins are indicated.

Figure 2.

Phenotypes of C. albicans ssn6 cells. (A) Mixed colony phenotypes of a freshly generated ssn6/ssn6 mutant (SGC123) restreaked on YPDA. (B) Characterization the various ssn6 colony types. Genotypes were rechecked by PCR diagnosis: +, wild-type allele; -, null allele (CAI8, SGC121 and SGC123: Table 1). For each colony type, cell morphology and growth in liquid medium in YPD at 30°C (length of lag phase, growth rate, and final OD₆₀₀) were determined. Growth on nonfermentable carbon sources as sole carbon source was examined on YNB plates at 30°C: +, robust growth; +/-, weak growth; -/+, very weak growth; -, no growth. (C) Morphogenetic responses of ssn6 (SGC123) and ssn6, cph1, efg1 cells (SGC133) after 3 h in YPD containing 10% serum.

Figure 3.

Phenotypic instability of C. albicans ssn6 cells. (A) The phenotypic stability of each colony type was assayed by replating cells on fresh YPD and measuring the proportion of colonies that had switched to an alternative form. (B) Phenotypic instability could be induced in a conditional ssn6/MET3-SSN6 strain following the addition of 2.5 mM methionine and cysteine to the medium: SSN6, CAI8; MET3-SSN6, SGC179 (Table 1).

Figure 4.

Phenotypic differences between wild-type, nrg1, tup1, and ssn6 cells. (A) Morphology of cells growing exponentially in YPD at 30°C: wild-type, CAI8 containing CIp10; nrg1, MMC3; tup1, BCA2-9; ssn6, SGC123 (Table 1). (B) Colony morphology (prewash) and invasiveness (postwash) of the same strains growing on YPD at 30°C. To measure invasiveness, cells were washed off the surface of the plate into H₂O with a glass spreader.

Figure 5.

(A) Numbers of C. albicans genes that were up- or down-regulated twofold or more in smooth and wrinkly ssn6 cells (SGC123), relative to the wild-type control (CAF2-1). (B) Numbers of C. albicans genes that were significantly up- or down-regulated in wrinkly ssn6 (SGC123), tup1 (BCA2-9), and nrg1 cells (MMC3), relative to the wild-type control (CAF2-1). Significant changes of twofold or more were identified using SAM (FDR set at 10%) using data from three independent replicates for each cell type.

Figure 6.

(A) Northern analysis of C. albicans mRNAs in wild-type (CAI8), tup1 (BCA2-9), ssn6 (SGC123), and nrg1 cells (MMC3). (B) Chromatin immunoprecipitation revealing association of Ssn6 with the WH11 promoter: wild-type cells (CAI8), ssn6 cells (SGC123); PCR with genomic DNA template, Total DNA; reaction with preimmune serum, PREI; reactions with anti-Ssn6 serum.

Figure 7.

(A) An NRE imposes repression on a lacZ reporter in C. albicans in an Nrg1-, Ssn6-, and Tup1-dependent manner: wild-type, CAI4; nrg1, MMC4; tup1, BCA2-9; ssn6, SGC124 (Table 1); basal reporter lacking NRE, basal; NRE [(C₅T)₄] containing reporter. (B) In a C. albicans one-hybrid assay, a lexA-Nrg1 fusion imposes repression on a Lex Operator-lacZ reporter in an Ssn6- and Tup1-dependent manner: wild-type, CAI8; nrg1, MMC9; tup1, CRC004; ssn6, SGC124. Fold repression was measured by comparing β-galactosidase levels (Miller Units) for C. albicans cells expressing LexA with those expressing the LexA-Nrg1 fusion. Means and standard deviations from triplicate assays on three independent transformants are shown.

Figure 8.

Models illustrating Ssn6 function in C. albicans. (A) Ssn6 is not required for Tup1-mediated repression at some promoters, suggesting that Tup1 might interact directly with the DNA-binding protein (DBP). (B) Ssn6 is partially required for Tup1-mediated repression at some C. albicans promoters, suggesting that Ssn6 stabilizes Tup1 interactions with the DNA-binding protein. (C) Ssn6 is essential for Tup1-mediated repression at other promoters, suggesting that Tup1 depends on Ssn6 for its interaction with the DNA-binding protein. The DNA-binding protein might differ between these promoters, or alternatively differences in the sequence context of the Response Element (RE) might affect the Ssn6 dependency. (D) The repression of some C. albicans promoters by Ssn6 is not dependent on Tup1 (see text).