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Role of Plc1p in regulation of Mcm1p-dependent genes

Katarzyna Guzinska, Roger Varghese, Ales Vancura
DOI: http://dx.doi.org/10.1111/j.1574-6968.2009.01602.x 245-250 First published online: 1 June 2009

Abstract

In budding yeasts, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) and several inositol polyphosphate kinases represent a nuclear pathway for synthesis of inositol polyphosphates (InsPs), which are involved in several aspects of DNA and RNA metabolism, including transcriptional regulation. Plc1p-produced inositol trisphosphate (InsP3) is phosphorylated by Ipk2p/Arg82p to yield InsP4/InsP5. Ipk2p/Arg82p is also a component of ArgR-Mcm1p complex that regulates transcription of genes involved in arginine metabolism. The role of Ipk2p/Arg82p in this complex is to stabilize the essential MADS box protein Mcm1p. Consequently, ipk2Δ cells display reduced levels of Mcm1p and attenuated expression of Mcm1p-dependent genes. Because plc1Δ cells display aberrant expression of several groups of genes, including genes involved in stress response, the objective of this study was to determine whether Plc1p also affects expression of Mcm1p-dependent genes. Here we report that not only ipk2Δ, but also plc1Δ cells display decreased expression of Mcm1p-dependent genes. However, Plc1p is not involved in stabilization of Mcm1p and affects transcription of Mcm1p-dependent genes by a different mechanism, probably involving regulation of chromatin remodeling complexes.

Keywords
  • phospholipase C
  • MCM1
  • transcriptional regulation
  • inositol polyphosphates

Introduction

In the budding yeast Saccharomyces cerevisiae, phospholipase C (Plc1p encoded by PLC1 gene) and four inositol polyphosphate kinases (Ipk2p/Arg82p, Ipk1p, Kcs1p, and Vip1p) constitute a nuclear signaling pathway that is responsible for synthesis of inositol polyphosphates (InsPs) and affects transcriptional control (Odom et al., 2000), export of mRNA from the nucleus (York et al., 1999), homologous DNA recombination (Luo et al., 2002), cell death, and telomere length (Saiardi et al., 2005; York et al., 2005). Hydrolysis of phosphatidylinositol-4,5-bisphosphate by phospholipase C yields inositol trisphosphate (InsP3) and diacylglycerol, and is the only pathway for InsPs synthesis in budding yeast cells. Ipk2p/Arg82p is a dual specificity kinase that converts Plc1p-generated InsP3 into InsP5 via InsP4 (Odom et al., 2000). InsP4 and InsP5 are involved in transcription by regulating chromatin remodeling complexes (Shen et al., 2003; Steger et al., 2003). InsP5 is subsequently converted to InsP6 by Ipk1p. InsP6 is an effector molecule that regulates mRNA export from the nucleus (York et al., 1999). The mechanism involves binding of InsP6 by nuclear pore protein Gle1p and stimulation of RNA-dependent ATPase activity of Dbp1p, which is essential for nuclear mRNA export (Alcazar-Roman et al., 2006; Weirich et al., 2006). Kcs1p and Vip1p are InsP6 and InsP7 kinases responsible for synthesis of 5-PP-InsP5 and 4-PP-InsP5/6-PP-InsP5, respectively (Saiardi et al., 1999, 2000; Lee et al., 2007; Mulugu et al., 2007). Ultimately, Kc1p and Vip1p can produce InsP8 molecules 4,5-PP2-InsP4 and 5,6-PP2-InsP4. It appears that inositol pyrophosphates are required for number of cellular functions, including inhibition of Pho80p-Pho85p cyclin-CDK complex by the Pho81p inhibitor (Luo et al., 2002; Saiardi et al., 2005; York et al., 2005; Lee et al., 2007).

Ipk2p/Arg82p, together with Arg80p, Arg81p, and Mcm1p, is a component of the transcriptional complex ArgR-Mcm1 that regulates transcription of genes involved in arginine metabolism (El Bakkoury et al., 2000). When arginine is present, these four proteins repress synthesis of arginine biosynthetic enzymes and induce synthesis of catabolic enzymes. Arg80p and Arg81p are specific regulators of the arginine system, while Ipk2p/Arg82p and Mcm1p are global regulators involved in other processes as well (Dubois & Messenguy, 1991; Elble & Tye, 1991; Messenguy & Dubois, 1993). Arg81p is the sensor of arginine that interacts with the two MADS box proteins Arg80p and Mcm1p to form a complex at the promoters of arginine regulated genes (El Bakkoury et al., 2000; Messenguy & Dubois, 2003). Mcm1p is an essential protein that plays a role in transcription of genes involved in M/G1 and G2/M cell-cycle progression, mating, recombination, and stress tolerance (Kuo & Grayhack, 1994; Messenguy & Dubois, 2003). The role of Ipk2p/Arg82p in the regulation of arginine-responsive genes consists of binding and stabilizing both Arg80p and Mcm1p but does not involve its InsP3 kinase activity (Dubois et al., 2000). However, the kinase activity of Arg82p is required for proper expression of genes regulated by phosphate and nitrogen (El Alami et al., 2003).

Using genome-wide expression analysis, our laboratory found previously that not only ipk2Δ, but also plc1Δ cells display decreased expression of Mcm1-dependent genes (Demczuk et al., 2008). Hence, in this study our objective was to determine whether Plc1p in addition to Ipk2p is also required for stabilization of Mcm1p and thus for expression of Mcm1p-dependent genes. In this report, we demonstrate that Plc1p is required for expression of Mcm1-depended genes; however, it does not affect the stability of Mcm1p. Therefore, Plc1p affects expression of Mcm1-dependent genes by a different mechanism, probably by affecting the activity of chromatin remodeling complexes.

Materials and methods

Strains and media

All yeast strains are listed in Table 1. Standard genetic techniques were used to manipulate yeast strains and to introduce mutations from non-W303 strains into the W303 background (Sherman, 1991). Cells were grown in rich medium [Yeast peptone dextrose (YPD); 1% yeast extract, 2% Bacto peptone, 2% glucose] or under selection in synthetic complete medium (CSM) containing 2% glucose and, when appropriate, lacking specific nutrients in order to select for a plasmid or strain with a particular genotype. Meiosis was induced in diploid cells by incubation in 1% potassium acetate.

View this table:
Table 1

Yeast strains used in this study

StrainsGenotypesSources/references
W303-1aMATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 ssd1-d2 can1-100R. Rothstein
W303-1αMATαade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 ssd1-d2 can1-100R. Rothstein
HL1-1W303-1αplc1::URA3Lin et al. (2000)
HL1-3W303-1a plc1::URA3DeLillo et al. (2003)
AOY138W303-1αipk2::kanMXYork et al. (2005)
A0004W303-1αipk1::kanMXYork et al. (1999)
YSC1178-7502110MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 MCM1-TAP ::HIS3Open Biosystems
KG026W303-1a MCM1-TAP ::HIS3This study
KG002W303-1a plc1 ::URA3 MCM1-TAP ::HIS3This study
KG022W303-1a ipk2 ::kanMX MCM1-TAP ::HIS3This study

β-Galactosidase assays

Wild-type, plc1Δ, and ipk2Δ strains were transformed with the following plasmids: pFV55 (CLN3-LacZ), pFV56 (FAR1-LacZ), pFV57 (PIS1-LacZ), pFV58 (CDC6-LacZ), and pFV60 (PMA1-LacZ) (El Bakkoury et al., 2000). The transformants were grown under selection in CSM-Ura medium at 30 °C and subsequently diluted in YPD medium to an A600 nm of 0.2 and grown until the culture reached A600 nm∼1.0. Cells from 10 mL culture were harvested by centrifugation and resuspended in β-galactosidase breaking buffer (100 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, 20% glycerol) containing protease inhibitors (Roche; Complete protease inhibitors) and 2 mM phenylmethylsulfonyl fluoride (PMSF). Samples were disrupted by vortexing with glass beads, and 10–100 μL of the collected supernatant was added to 0.9 mL of Z buffer (60 mM Na2HPO4·7H2O; 40 mM NaH2PO4·H2O; 10 mM KCl; 1 mM MgSO4, 50 mM β-mercaptoethanol, pH 7.0). The volume was adjusted up to 1 mL with β-galactosidase breaking buffer and the assay mixture incubated in water bath at 28 °C for 5 min with moderate shaking. Reaction was initiated by adding 0.2 mL of o-nitrophenyl-β-d-galactopyranoside (ONPG; 4 mg mL−1 in Z buffer) and continued at 28 °C until mixture turned pale yellow and terminated by addition of 0.5 mL of 1 M Na2CO3 (Choi et al., 1998). Protein concentration was determined using the Coomassie Plus protein assay kit (Pierce). The specific activity was expressed in terms of nanomoles of ONPG hydrolyzed per minute per milligram of protein.

Real-time reverse transcriptase (RT)-PCR analysis

Total RNA was isolated from cultures grown in YPD medium to A600 nm∼1.0 by the hot phenol method as described previously (Iyer & Struhl, 1996), treated with RNAse-free DNAse (Qiagen), and purified with an RNeasy mini kit (Qiagen). RT and real-time PCR amplification using BioRad MyIQ single color real-time PCR detection system (Bio-Rad) were performed with iScript kit (Bio-Rad), 100 ng of RNA, and the following primers: ACT1 (5′-TATGTGTAAAGCCGGTTTTGC-3′ and 5′-GACAATACCGTGTTCAATTGGG-3′), STE2 (5′-ACCATCACTTTCGATGAGTTGC-3′ and 5′-GGTTGATAATGAAAATCGGCG-3′), STE3 (5′-CCTTTAGCAT GG CATTCACATAC-3′ and 5′-GATATGCCAATATTCGCACCAAC-3′), BAR1 (5′-ACGAAGAGGAGATGTATTACGCAAC-3′ and 5′-ACCTGCAATCAATTGAAGGC-3′), and MFα1 (5′-GCTGAAGCTGTCATCGGTTACTTAG-3′ and CCAGGTTTT AGTTGCAACCAATG-3′).

Western blotting

Yeast cultures were grown in 200-mL YPD to an A600nm=1.0. To determine the Mcm1p stability, 20 μg mL−1 cycloheximide was added at time zero. At different time points after the treatment, cells from 30 mL of the culture were harvested by centrifugation and subsequently converted to spheroplasts using yeast lytic enzyme (Sigma; 700 U mL−1 in 10 mM Tris-HCl buffer, pH 7.5, containing10% sucrose). Protein concentration of the samples was determined using Coomassie Plus protein assay kit (Pierce). Denatured proteins were separated on 10% denaturing polyacrylamide gel and Western blotting with 0.5 μg mL−1 of anti-TAP antibody (Open Biosystems) was carried out as described previously (Demczuk et al., 2008) and the blots were visualized using GeneGnome Bioimaging System (Syngene). To confirm equivalent amounts of loaded proteins, the membrane was stripped and incubated with anti-3-phosphoglycerate kinase antibody (Molecular Probes).

Results and discussion

To identify transcriptional targets of InPs, our laboratory performed genome-wide expression analysis with wild-type, plc1Δ, ipk2, and ipk1Δ strains (Demczuk et al., 2008). In addition to increased expression of Msn2p-dependent stress-responsive genes in plc1Δ cells, we also observed decreased expression of cell-type-specific and Mcm1p-dependent genes such as BAR1, MFA1, and STE2 in ipk2Δ strain (Demczuk et al., 2008). This was not entirely surprising, because ipk2Δ cells display decreased stability of Mcm1p (El Bakkoury et al., 2000). However, several of the Mcm1p-dependent genes were expressed at a lower level also in the plc1Δ cells (Demczuk et al., 2008). These results suggested that Plc1p and/or synthesis of InsPs may be required for full expression of Mcm1p-dependent genes, perhaps also by affecting the stability of Mcm1p. One possible model that could account for the stabilizing effect of InsP3 on Mcm1p would involve assumption that Ipk2p/Arg82 must bind InsPs in order to stabilize Mcm1p.

To analyze the involvement of Plc1p and InsP3 in the control of Mcm1-dependent genes, wild-type, plc1Δ, and ipk2Δ strains were transformed with plasmids containing lacZ reporter gene under the control of Mcm1p-regulated promoters of CLN3, FAR1, PIS1, CDC6, and PMA1 (El Bakkoury et al., 2000). As reported previously (El Bakkoury et al., 2000), β-galactosidase activities were strongly reduced in ipk2Δ strain in comparison with wild type strain. In plc1Δ strain the activities were also reduced, however, to a lesser extent than in the ipk2Δ strain (Fig. 1). These results thus suggest that Plc1p is also required for full expression of Mcm1p-dependent genes; however, Plc1p appears to be less important than Ipk2p.

Figure 1

plc1Δ cells display reduced expression of Mcm1p-dependent genes. Wild-type, plc1Δ, and ipk2Δ strains were transformed with plasmids containing lacZ reporter genes under the control of the indicated Mcm1p-regulated promoters (El Bakkoury et al., 2000). The β-galactosidase assays were carried out as described previously (Choi et al., 1998), and the values were calculated from three independent experiments and represent means±SD.

Because the expression of Mcm1p-dependent genes might be affected by promoter chromatin structure that may not be faithfully reconstituted in the context of reporter plasmids, we determined expression of several Mcm1p-dependent genes in their normal chromosomal locations. RNA was isolated from both MATa and MATα cells of wild-type, plc1Δ, and ipk2Δ strains, and the relative transcript levels of a-specific genes (BAR1, STE2) and α-specific genes (STE3 and MFα1) were determined. In MATα cells, cell-type specific transcriptional factor Matα1p and Mcm1p bind cooperatively to promoters of corresponding genes, thus activating α-cell-type-specific gene expression. In a cells, Mcm1p activates transcription of a-specific genes by binding to the Mcm1-binding site found in promoter regions of a-specific genes. Thus, expression of α-specific genes in MATα cells as well as expression of a-specific genes in MATa cells requires Mcm1p (Hwang-Shum et al., 1991; Mead et al., 2002). Previous studies have shown that Ipk2p is required for the expression of certain a- and α-specific genes that are also controlled by Mcm1p (Dubois & Messenguy, 1994). As shown in Fig. 2a, the transcription levels of a – specific genes BAR1 and STE2 in ipk2Δ mutant were significantly lower than those found in wild-type cells. The same pattern was observed in plc1Δ strain, however, the decrease was not as dramatic as in ipk2Δ strain (Fig. 2a). The transcript levels of α-specific genes STE3 and MFα1 in ipk2Δ strain were also significantly reduced as compared with wild type. Again, the pattern observed in plc1Δ strain was similar but the decrease was not as dramatic as in ipk2Δ strain (Fig. 2b).

Figure 2

plc1Δ cells display reduced expression of cell-type-specific genes. The indicated strains were grown in YPD medium at 30°C to A600 nm=1.0 and the total RNA was isolated and assayed for ACT1, BAR1, STE2, STE3, and MFα1 transcripts by real time RT-PCR. The results were normalized to ACT1 RNA and expressed as relative values in comparison with corresponding WT strain. The experiment was repeated three times and the results represent means±SD. (a) Expression of α-specific genes BAR1 and STE2 in Mata cells. (b) Expression of α-specific genes STE3 and MFα1 in MATα cells.

The above results support the conclusion that not only Ipk2p, but also Plc1p is important for full expression of Mcm1p-dependent genes. However, the ipk2Δ mutation affects expression of Mcm1p-dependent genes more significantly than plc1Δ mutation. Because Ipk2p affects expression of Mcm1p-dependent genes by physically interacting with and stabilizing Mcm1p (El Bakkoury et al., 2000; El Alami et al., 2003), we considered possibility that Plc1p also affects intracellular level of Mcm1p. There are several indications that Plc1p may be involved in regulation of stability of certain proteins. First, 26S proteasome-mediated destruction of C-type cyclin Ume3p/Srb11p/Ssn3p upon oxidative stress requires Plc1p (Cooper et al., 1999). Second, genome-wide identification of protein complexes revealed that Plc1p interacts with Caf130p (Krogan et al., 2006), a component of Ccr4/Not transcriptional regulatory complex. One of the subunits of the Ccr4-Not complex is Not4p, a ubiquitin E3 ligase (Albert et al., 2002; Collart, 2003) that interacts with Ubc4p, another ubiquitin-conjugating enzyme. In addition, the Ccr4-Not complex associates with the proteasome (Laribee et al., 2007). These findings suggest that at least fraction of Plc1p is found in a molecular complex with ubiquitin-conjugating enzymes and proteasome. In addition, we have described previously that Plc1p is required for recruitment of histone aceryltransferase complex SAGA to Sko1p-regulated promoters (Guha et al., 2007). Coincidentally, the proteasome 19S regulatory particle was found to facilitate loading of SAGA onto chromatin (Lee et al., 2005). These findings prompted us to test whether Plc1p, similarly to Ipk2p, affects stability of Mcm1p. We examined stability of TAP-tagged Mcm1p in WT, plc1Δ, and ipk2Δ strains. The cells were grown in YPD medium to early exponential phase and subsequently treated with cycloheximide to block protein synthesis. Mcm1p levels were determined by Western blotting using anti-TAP antibodies. In comparison with wild-type cells, the amount of Mcm1p in ipk2Δ strains is reduced to about 60% before treatment with cycloheximide and to about 20% after 3-h treatment with cycloheximide (Fig. 3). This result demonstrates that Mcm1p is less stable in ipk2Δ strain and agrees with previous results that showed that Ipk2p is required for Mcm1p stability (El Bakkoury et al., 2000). In contrast, 3-h treatment with cycloheximide in wild-type as well as plc1Δ cells reduced Mcm1p level only to 90% (Fig. 3). Thus, Plc1p is not required for Mcm1p stability and Plc1p and/or synthesis of InsP3 affect expression of Mcm1p-dependent genes by a different mechanism. Because InsPs regulate promoter recruitment and activity of chromatin remodeling complexes such as Swi/Snf and Ino80 (Shen et al., 2003; Steger et al., 2003), it is likely that reduced recruitment and/or activity of chromatin remodeling complexes in plc1Δ cells is responsible for decreased expression of Mcm1p-dependent genes. Future experiments will address the role of InsPs in regulation of chromatin remodeling complexes and expression of Mcm1p-dependent genes.

Figure 3

Plc1p is not required for Mcm1p stability. (a) Wild-type, ipk2Δ, and plc1Δ cells expressing TAP-tagged Mcm1p were grown in YPD medium to early exponential phase (A600 nm=1.0) and subsequently treated with cycloheximide to block protein synthesis. Mcm1p-TAP levels were determined by Western blotting using anti-TAP antibody before addition of cycloheximide and after 1 and 3 h. Even loading of protein samples was confirmed with anti-3-phosphoglycerate (Pgk1p) antibody. The experiment was performed three times, and representative results are shown. (b) Densitometric evaluation of the representative Western blot.

Acknowledgements

We thank Drs Messenguy, Wente, and York for strains and plasmids and members of Vancura lab for helpful comments. This work was supported by grants from the National Institutes of Health (GM076075) to A.V.

Footnotes

  • Editor: Linda Bisson

References

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