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. 2013 Sep;12(9):1293-304.
doi: 10.1128/EC.00137-13. Epub 2013 Jul 19.

Differential regulation of white-opaque switching by individual subunits of Candida albicans mediator

Anda Zhang  1 , Zhongle Liu, Lawrence C Myers
Affiliations

Differential regulation of white-opaque switching by individual subunits of Candida albicans mediator

Anda Zhang et al. Eukaryot Cell. 2013 Sep.

Abstract

The multisubunit eukaryotic Mediator complex integrates diverse positive and negative gene regulatory signals and transmits them to the core transcription machinery. Mutations in individual subunits within the complex can lead to decreased or increased transcription of certain subsets of genes, which are highly specific to the mutated subunit. Recent studies suggest a role for Mediator in epigenetic silencing. Using white-opaque morphological switching in Candida albicans as a model, we have shown that Mediator is required for the stability of both the epigenetic silenced (white) and active (opaque) states of the bistable transcription circuit driven by the master regulator Wor1. Individual deletions of eight C. albicans Mediator subunits have shown that different Mediator subunits have dramatically diverse effects on the directionality, frequency, and environmental induction of epigenetic switching. Among the Mediator deletion mutants analyzed, only Med12 has a steady-state transcriptional effect on the components of the Wor1 circuit that clearly corresponds to its effect on switching. The MED16 and MED9 genes have been found to be among a small subset of genes that are required for the stability of both the white and opaque states. Deletion of the Med3 subunit completely destabilizes the opaque state, even though the Wor1 transcription circuit is intact and can be driven by ectopic expression of Wor1. The highly impaired ability of the med3 deletion mutant to mate, even when Wor1 expression is ectopically induced, reveals that the activation of the Wor1 circuit can be decoupled from the opaque state and one of its primary biological consequences.

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Figures

Fig 1
Fig 1
Graphical summary of C. albicans Mediator subunit architecture and the impact of deletion mutants on white (W)-opaque (O) switching as described in detail in Table 1. The architecture of the C. albicans Tail module is based on published data (13) and data presented here, while the remainder is modeled on the structure of the S. cerevisiae complex (9).
Fig 2
Fig 2
The Med2(Tlo)/Med3/Med15 Tail submodule of caMediator remains associated with the Middle and Head modules in a med16Δ/Δ strain. (A) Silver stain gels of Mediator purified from WT (Med8-6×His-Flag, Med3-3XHA) (lane 1), med16Δ/Δ (Med8-6×His-Flag, Med3-3XHA) (lane 2), and med16Δ/Δ (Med15-6×His-Flag, Med3-3XHA) (lane 3) strains. To obtain adequate resolution over the entire molecular mass range, we show panels from 6%, 7.5%, and 12.5% SDS-PAGE from top to bottom. (B) Western blot of the three samples analyzed as described for panel A, showing equal recoveries of Med3-HA (anti-HA) in WT and mutant Mediator using purification tags on Med8 or Med15. An antibody against caMed1 served as the recovery control.
Fig 3
Fig 3
Kinetics of temperature-induced opaque-white switching in Mediator mutant strains. Opaque cells of WT and Mediator mutant strains growing in SC+Glu media, shifted from 25°C to 37°C, were removed every 1.5 h for analysis. The percentage of opaque cells that had switched to white (y axis) was calculated as the ratio of the fraction of opaque cells at each time point relative to the fraction of opaque cells at the starting time point (0 h), which was usually close to 100%. Data were plotted as means ± standard deviations (SD) of the results of three independent experiments with more than 1,500 colonies counted in total for each strain.
Fig 4
Fig 4
Steady-state mRNA and protein levels of key transcriptional regulators of white-opaque switching. (A) RT-qPCR analysis of the mRNA levels of WOR1, EFG1, WOR2, and CZF1 in white and opaque cells growing in SC+Glu media. The qPCR was performed in triplicate. Results were normalized to ACT1. The amount of transcript in each mutant was compared to that in the WT white or opaque cells using a t test. Bars represent means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Immunoblotting shows that the Wor1 protein level increased in med3Δ/Δ mutant strain white cells relative to the level in WT and other Mediator mutant cells. Tubulin was used as a loading control. (C) Diagram of the interlocking loop that regulates white-opaque switching (modified from PLOS Biology [41]). Dashed lines indicate regulatory controls active in the white state, and solid lines indicate regulatory controls active in the opaque state.
Fig 5
Fig 5
Ectopic Wor1 expression activated the Wor1 circuit and altered morphologies in med3Δ/Δ and med12Δ/Δ mutants. (A) RT-qPCR analysis shows that ectopically expressed WOR1 induces endogenous WOR1 expression in both med3Δ/Δ and med12Δ/Δ mutants. Endogenous WOR1 was measured by primers targeting the 5′UTR of endogenous WOR1 (based on RNA-seq data from reference 63). Ectopic WOR1 was measured by primers targeting the end of the WOR1 ORF in junction with 3HA. The qPCR was performed in triplicate. Results were normalized to ACT1. Bars represent means ± SD. *, P < 0.05 (t test for the comparison between WT and med3Δ/Δ in SC+Glu); **, P < 0.01 (t test for comparisons between WT and med3Δ/Δ and med12Δ/Δ in SC+Mal). (B) Immunoblotting using an anti-HA antibody showed that ectopic Wor1-3HA protein expression was detected at similar levels in med3Δ/Δ and med12Δ/Δ mutants compared with the WT; however, densitometry of the Wor1 band (α-Wor1) band under conditions of ectopic Wor1 expression showed that the total amount of Wor1 was reduced by ∼50% in the med3Δ/Δ and med12Δ/Δ strains compared to the WT. An antibody against tubulin was used as a loading control. (C) A med3Δ/Δ mutant with ectopic Wor1 expression formed an altered opaque colony morphology. Cells were preincubated on either SC+Glu or SC+Mal plates for 3 days and replated onto phloxine B plates. Photos were taken with a Nikon SMZ1500 stereomicroscope (Nikon Instruments). (D) Measurement of cell dimensions showed that ectopic Wor1 expression induced altered cell morphology in a med3Δ/Δ mutant. The percentage of elongated cells (length/width > 2) was significantly less in the med3Δ/Δ mutant than in the WT and complemented strains. Cells were harvested from SC+Glucose (WT Whi and Opa, OFF) or SC+Mal (ON) liquid culture for microscopic analysis. A minimum of 50 cells was observed for each morphology evaluation. l, length; w, width (maximum diameter) (40). For representative figures of individual cell morphologies, see Fig. S2 in the supplemental material.
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