Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast

Abstract

Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca2+ signaling. We describe new components of a calcineurin-mediated response in yeast, the Ca2+-induced transcriptional activation of FKS2, which encodes a beta-1,3 glucan synthase. A 24-bp region of the FKS2 promoter was defined as sufficient to confer calcineurin-dependent transcriptional induction on a minimal promoter in response to Ca2+ and was named CDRE (for calcineurin-dependent response element). The product of CRZ1 (YNL027w) was identified as an activator of CDRE-driven transcription. Crz1p contains zinc finger motifs and binds specifically to the CDRE. Genetic analysis revealed that crz1Delta mutant cells exhibit several phenotypes similar to those of calcineurin mutants and that overexpression of CRZ1 in calcineurin mutants suppressed these phenotypes. These results suggest that Crz1p functions downstream of calcineurin to effect multiple calcineurin-dependent responses. Moreover, the calcineurin-dependent transcriptional induction of FKS2 in response to Ca2+, alpha-factor, and Na+ was found to require CRZ1. In addition, we found that the calcineurin-dependent transcriptional regulation of PMR2 and PMC1 required CRZ1. However, transcription of PMR2 and PMC1 was activated by only a subset of the treatments that activated FKS2 transcription. Thus, in response to multiple signals, calcineurin acts through the Crz1p transcription factor to differentially regulate the expression of several target genes in yeast.

Figure 1

Constructs containing 762 bp or more of FKS2 upstream sequence can support calcineurin-dependent transcriptional induction of expression. Plasmid-based lacZ reporter genes were constructed with varying amounts of FKS2 upstream sequence [−910/+6 (pAMS312), −762/+6 (pAMS317), −705/+6 (pAMS319)]. β-Galactosidase activities are shown for extracts from cells (YPH499) that were either untreated (open bar), treated with 10 μg/ml of α-factor (shaded bar), or treated with 200 mm CaCl₂ (solid bar) in the presence or absence of FK506. Each extract was assayed in triplicate, and the s.d. is representative of the error between these samples.

Figure 2

Tandem copies of CDRE increase the response of heterologous promoters to Ca²⁺ and α-factor. Plasmid-based lacZ reporter genes were constructed containing heterologous promoters with increasing copies of the CDRE or mutCDRE in tandem [1×-CDRE (pAMS342), 2×-CDRE (pAMS363), 4×-CDRE (pAMS366), 4×-mutCDRE (pAMS364)]. β-Galactosidase activities are shown for extracts from cells (YPH499) that were either untreated (open bar), treated with 10 μg/ml of α-factor (shaded bar), or treated with 200 mm CaCl₂ (solid bar) in the presence or absence of FK506. Each extract was assayed in triplicate and the s.d. is representative of the error between these samples. (Note discontinuity in y-axis.)

Figure 3

CRZ1 encodes a putative transcription factor. The predicted amino acid sequence of the transcription factor Crz1p (ORF YNL027w) is shown with the polyglutamine tract at the amino terminus underlined and the three zinc fingers at the carboxyl terminus boxed in gray. Conserved cysteine and histidine residues within each zinc finger are indicated by a mark (▾) above each residue.

Figure 4

Crz1p binds to the CDRE. Extracts of strains YPH499 containing YEp351 (“WT”), ASY472 containing YEp351 (“crz1Δ”), and ASY472 containing pAMS446 [“crz1Δ, YEp(HA–CRZ1)”] were analyzed by DNA mobility retardation analysis using the CDRE as probe (see Materials and Methods). The plasmid YEp(HA–CRZ1) carries a gene encoding a version of Crz1p with the HA epitope inserted near the amino terminus of the protein. (Lanes 5–7) Equal amounts of α-HA antibody, α-myc antibody, or antibody dilution buffer were added 5 min before gel loading; (lanes 8,9) a 100-fold molar excess of unlabeled CDRE or mut-CDRE oligonucleotide to probe was added before addition of extract.

Figure 5

CRZ1 mutants exhibit defects similar to but less severe than those of calcineurin mutants. (A) Saturated overnight cultures were diluted, spread on YPD media containing the specified ions (1.8 mm MnCl₂ or 0.16 m LiCl), and grown for 2–5 days at 30°C (Pozos et al. 1996). Strains are YPH499 (“WT”), ASY472 (“crz1Δ”), DD12 (“cnb1Δ”), and ASY475 (“cnb1Δ crz1Δ”). (B) Cells were grown on low pH YPD media with or without the indicated concentrations of α-factor. The fraction viable was determined by comparing growth on plates containing α-factor with growth on plates without α-factor (Moser et al. 1996). Each data point represents an average of three plates. Strains are YPH499 (▪), ASY472 (□), DD12 (▴), and ASY475 (▵).

Figure 6

Increased expression of CRZ1 suppresses calcineurin mutant phenotypes. (A) Saturated overnight cultures were diluted and spread on synthetic media with or without ions (5 mm MnCl₂ or 0.55 m LiCl) and grown for 2–5 days at 30°C (Pozos et al. 1996). Strains are YPH499 containing YEp351 (“WT + YEp351”), DD12 containing YEp351 (“cnb1Δ + YEp351”), and DD12 containing pAMS435 (“cnb1Δ + YEpCRZ1”). (B) Cells were grown on synthetic media with or without the indicated levels of α-factor. The fraction viable was determined by comparing growth on plates containing α-factor with growth on plates without α-factor (Moser et al. 1996). Each data point represents an average of three plates. Strains are YPH499 containing YEp351 (WT + YEp351, ▪), YPH499 containing pAMS435 (WT + YEpCRZ1, □), DD12 containing YEp351 (cnb1Δ + YEp351, ▴), and DD12 containing pAMS435 (cnb1Δ + YEpCRZ1, ▵).

Figure 7

CRZ1 is necessary for calcineurin-dependent transcriptional induction of the FKS2 gene in vivo. YPH499 (“WT”) and ASY472 (“crz1Δ”) cells were incubated for 4 hr in low pH YPD with or without FK506 and inducing treatments. Total RNA isolated from these cells was subjected to Northern blot and hybridized successively to FKS2 and ACT1 probes. FKS2 mRNA levels were normalized to ACT1 mRNA levels and are from one representative experiment with mRNA from cells that were either untreated (open bars) or treated with 10 μg/ml of α-factor (shaded bars), 0.8 m NaCl (hatched bars), or 200 mm CaCl₂ (solid bars). The ACT1-normalized FKS2 mRNA level in untreated wild-type cells was arbitrarily designated a value of 1.

Figure 8

CRZ1 is a general mediator of calcineurin-dependent transcriptional regulation. (A) Wild-type cells containing a plasmid-based pmr2::lacZ reporter gene (pFR70; Marquez and Serrano 1996) were grown at 30°C for 4 hr with or without FK506 in either YPD (open bar), synthetic media containing 200 mm CaCl₂ (solid bar), or YPD containing 0.8 m NaCl (hatched bar). (B) Wild-type cells containing a plasmid-based pmc1::cyc1::lacZ reporter gene (pAMS381) were grown at 30°C for 4 hr with or without FK506 in either YPD (open bar), low pH YPD containing 200 mm CaCl₂ (solid bar), or YPD containing 0.8 m NaCl (hatched bar). β-Galactosidase activities are shown for extracts from cells (YPH499) from one representative experiment. Each extract was assayed in triplicate, and the s.d. is representative of the error between these samples.

Figure 9

Model describing calcineurin-mediated signal transduction pathways. (See Discussion for description.)