Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae - PubMed (original) (raw)

Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae

D P Matheos et al. Genes Dev. 1997.

Abstract

Ca2+ signals regulate gene expression in animal and yeast cells through mechanisms involving calcineurin, a protein phosphatase activated by binding Ca2+ and calmodulin. Tcn1p, also named Crz1p, was identified as a transcription factor in yeast required for the calcineurin-dependent induction of PMC1, PMR1, PMR2A, and FKS2 which confer tolerance to high Ca2+, Mn2+, Na+, and cell wall damage, respectively. Tcn1p was not required for other calcineurin-dependent processes, such as inhibition of a vacuolar H+/Ca2+ exchanger and inhibition of a pheromone-stimulated Ca2+ uptake system, suggesting that Tcn1p functions downstream of calcineurin on a branch of the calcium signaling pathway leading to gene expression. Tcn1p contains three zinc finger motifs at its carboxyl terminus resembling the DNA-binding domains of Zif268, Swi5p, and other transcription factors. When fused to the transcription activation domain of Gal4p, the carboxy terminal domain of Tcn1p directed strong calcineurin-independent expression of PMC1-lacZ and other target genes. The amino-terminal domain of Tcn1p was found to function as a calcineurin-dependent transcription activation domain when fused to the DNA-binding domain of Gal4p. This amino-terminal domain also formed Ca2+-dependent and FK506-sensitive interactions with calcineurin in the yeast two-hybrid assay. These findings suggest that Tcn1p functions as a calcineurin-dependent transcription factor. Interestingly, induction of Tcn1p-dependent genes was found to be differentially controlled in response to physiological Ca2+ signals generated by treatment with mating pheromone and high salt. We propose that different promoters are sensitive to variations in the strength of Ca2+ signals generated by these stimuli and to effects of other signaling pathways.

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Figures

Figure 1

Figure 1

Treatments with CaCl2, pheromone, or high salt generate Ca2+ signals that differentially induce calcineurin-dependent reporter genes. Wild-type yeast (strain W303-1A) was transformed with either plasmid pKC190 carrying PMC1–lacZ (A), plasmid pKC201 carrying PMR2A–lacZ (B), or plasmid pDM5 containing FKS2–lacZ (C), grown to mid-log phase and treated for 4 hr at 30°C in YPD (pH 5.5) medium with 0.2 μg/ml of FK506 (solid bars) or without FK506 (shaded bars) with the additional supplements of Ca2+ (100 m

m

CaCl2), pheromone (20 μg/ml), Na+ (750 m

m

NaCl), or combinations thereof as indicated at the base of the plot. Each bar represents the average of three independent determinations of accumulated β-galactosidase activity (±

s.d.

).

Figure 2

Figure 2

Sequence features of Tcn1p. (A) Predicted open reading frame of TCN1. Denoted are putative domains of Tcn1p: three acidic regions with net charges of −7, −23, and −10, respectively; a Q-rich domain where 24 of 27 amino acid residues are glutamine; a basic region containing a net charge of +13; and three putative zinc fingers. The arrow indicates the division between the amino and carboxyl termini used to assay functionality of these two domains. (B) Multiple sequence alignment of the three zinc finger motifs from Tcn1p, and the transcription factors Swi5p (residues 550–632) from yeast (Stillman et al. 1988) and Zif268/EGR-1 (residues 287–367) from mammals (Lemaire et al. 1988). Residues conserved in at least two of the three sequences are boxed and highlighted. Residues that coordinate zinc ions (asterisks) and that contact DNA (∧) in the crystal structure of Zif268 complexed with DNA (Pavletich and Pabo 1991) are indicated.

Figure 3

Figure 3

Functional domains of Tcn1p defined by fusions with Gal4p. (A) Expression of a PMC1–lacZ reporter gene on plasmid pKC190 in a tcn1 null mutant (strain DMY14) driven by either a Tcn1(C)::Gal4(AD) hybrid protein (plasmid pDM16) or a Gal4(AD) fragment (plasmid pPC86) as a control was measured after 4 hr growth in YPD (pH 5.5) medium supplemented as indicated with 200 m

m

CaCl2 and 0.2 μg/ml of FK506. Data are the averages of three independent transformants with standard deviation as indicated by error bars. Similar results were obtained using PMR2A–lacZ (pKC201) and FKS2–lacZ (pDM5) reporter genes. (B–D) Expression of a GAL1–lacZ reporter gene in a gal4 gal80 double mutant Y190 (Harper et al. 1993) was measured as above using plasmids expressing the following hybrid proteins: (B) Gal4(DB)::Tcn1(N) on plasmid pDM15 or Gal4(DB) on plasmid pPC97; (C) Tcn1(N)::Gal4(AD) on plasmid pTJK27 with either Gal4(DB) on plasmid pPC97 or Gal4(DB)::Cna1ΔC on plasmid pKC116; (D) Gal4(DB)::Cna1 on pKC115 with either Tcn1(N)::Gal4(AD) on plasmid pTJK27 or Gal4(AD) on plasmid pPC86. The results show the carboxyl terminus of Tcn1p interacts functionally with the promoter of PMC1 (A), whereas the amino-terminal region of Tcn1p functions as both a calcineurin-dependent transcription activation domain (B) and a calcineurin-interacting domain (C,D).

Figure 4

Figure 4

Mn2+, Na+, and Ca2+ tolerance assays of various yeast mutants showing roles of Tcn1p. All strains were grown to saturation in YPD medium at 30°C and diluted 1000-fold into fresh media containing a range of MnCl2, NaCl, or CaCl2 concentrations (with and without 0.2 μg/ml of FK506) and incubated for 1 day at 30°C in flat-bottom 96-well dishes (0.2 ml/well). Optical density at 650 nm was measured for each resuspended culture and plotted directly (A,B) or plotted and used to determine the 50% inhibitory concentration or IC50 (C) as described in Materials and Methods.

Figure 5

Figure 5

Tcn1p is not required for calcineurin-dependent inhibition of Ca2+ uptake stimulated by pheromone. Log-phase cells were incubated for 4 hr at 30°C in YPD medium supplemented with 45Ca2+ tracer in the presence or absence of synthetic pheromone [10 μ

m

of α-mating factor (MF)] and FK506 (1.0 μg/ml). Total cell-associated Ca2+ was determined as described in Materials and Methods, and the average of three independent experiments are shown (±

s.d.

). A large stimulatory effect of FK506 was observed for both the wild-type (strain W303-1A) and tcn1 null mutant (strain DMY14).

Figure 6

Figure 6

Calcineurin-dependent induction of TCN1–lacZ and other reporter genes is enhanced by overexpression of Tcn1p. Wild-type yeast (strain W303-1A) carrying the indicated reporter genes (plasmids pDM7, pKC190, pDM5, and pKC201, respectively) were transformed with either a control plasmid (YEp13, ○) or a similar high dosage plasmid containing TCN1 (pLE66, •) and grown to log phase in SC − ura − leu medium to maintain plasmid selection. After incubation for 4 hr at 30°C in YPD (pH 5.5) medium supplemented with CaCl2 as indicated and either with FK506 (0.4 μg/ml, broken lines) or without FK506 (solid lines), cells were collected and assayed for β-galactosidase accumulation.

Figure 7

Figure 7

Differential expression of Tcn1p-dependent genes in response to strength of Ca2+ signals and other regulatory inputs. (A) Wild-type strain W303-1A was transformed with both plasmid pKC190 carrying PMC1–lacZ and plasmid pLE66 to increase dosage of Tcn1p, grown, and assayed for β-galactosidase activity as described in Fig. 1. (B) The MATa gal4 gal80 strain Y190 containing a GAL1–lacZ reporter gene was transformed with plasmid pDM15 expressing the Gal4(DB)::Tcn1(N) hybrid factor and then grown and assayed as above. The results suggest pheromone produces a weak Ca2+ signal that partially induces a high sensitivity reporter (B) but fails to induce a low sensitivity reporter unless Tcn1p is overexpressed (cf. A with Fig. 1A). High salt treatment induces the high sensitivity reporter and prevents induction of PMC1–lacZ by high Ca2+ treatment despite overexpression of Tcn1p.

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