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The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 → S transition
Biochemical and Biophysical Research Communications 410 (2011) 276–281
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The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 ? S transition Pooja Aggarwal 1, Bhavna Padmanabhan, Abhay Bhat, Kavitha Sarvepalli, Parag P. Sadhale, Utpal Nath ⇑ Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
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Article history: Received 21 May 2011 Available online 30 May 2011 Keywords: TCP transcription factor Arabidopsis Cell cycle Budding yeast G1 ? S transition
a b s t r a c t The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 ? S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 ? S arrest is discussed. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction TCP proteins are plant-specific transcription factors that control diverse functions in plants [1–6]. They share a conserved DNAbinding domain, known as TCP domain, that folds into basic helix–loop–helix structure. TCP proteins are classified into two groups; class I and II [7,8]. Several studies indicate that class I TCP proteins promote cell division whereas class II proteins repress organ growth by inhibiting cell proliferation [9]. For example, class I TCP proteins PCF1 and PCF2 in rice bind to the PCNA promoter [10] and TCP20 in Arabidopsis interacts with the cis-element of cyclin B [11]. However, a direct link between class II TCP proteins and cell cycle genes has not been demonstrated. CINCINNATA (CIN) in Antirrhinum and TCP4 in Arabidopsis are homologous class II TCP proteins that control leaf morphogenesis by repressing cell division [3,4]. Although it has been postulated that CIN alters the transcriptional status of cell cycle genes [3], its exact mechanism is poorly understood. When multiple CIN-like TCP genes were mutated in Arabidopsis, the mitotic index in developing leaves was not significantly altered [6], suggesting an indirect role for CIN-like TCP proteins in controlling cell cycle. To investigate the effect of TCP proteins on cell cycle more objectively and in greater detail, we have studied their effect on cell division in budding yeast, an organism that lacks TCP-like genes.
⇑ Corresponding author. E-mail address: [email protected] (U. Nath). Present address: Temasek Life Sciences Laboratory Limited, 1 Research Link, National University of Singapore, Singapore 117604, Singapore. 1
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.132
Cell cycle is remarkably conserved in yeast, animals, and plants and is controlled by hetero-dimeric CDK (cyclin-dependent kinases)/cyclin complexes [12]. Three major checkpoints control the execution of cell cycle: G1 ? S, G2 ? M and metaphase ? anaphase transitions. Budding yeast has a single CDK, Cdc28, that regulates progression through all phases of cell cycle in association with different cyclin partners, such as Cln1–3 (G1 cyclin), Clb5–6 (S cyclin) and Clb1–4 (M cyclin) [13]. Arabidopsis has several classes of CDKs (CDKA-F) and more than 49 cyclins [14]. A & B classes of CDKs are the two major drivers of plant cell cycle. CDKAs share homology with yeast and mammalian CDKs and control both G1–S and G2–M transitions. CDKBs are specific to plants and are restricted to G2–M phase. Among several classes of cyclins identified in plants, CYCA, CYCB and CYCD constitute three major groups. CYCD, in association with CDKA, regulates G1 ? S transition. Activity of CDK–cyclin complexes is further regulated by proteins such as CDK-activating enzymes (CAK) and CDK inhibitory proteins (CKIs) [15,16]. Regulation of cell cycle by CKIs is conserved in yeast, animals and plants. These proteins act as the regulators of G1 ? S checkpoint in response to environmental and developmental cues and thus orchestrate eukaryotic cell proliferation and differentiation [16]. In budding yeast, the major CKI, Sic1, prevents premature S phase initiation until Cdc28/Cln1–2 levels are sufficient to complete G1 phase successfully [17]. Direct effect of various genes on the plant cell-cycle regulation is more difficult to study mainly due to the complexity of the core cell cycle components in plants. Conservation of the basic tools of the cell cycle machinery among plants and yeast has, however, enabled the isolation of many plant CDKs and cyclins by comparative
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genomics. For instance, genetic complementation of G1 cyclindeficient yeast mutant cells has led to the cloning of cyclin D in plants [18], suggesting that yeast can be successfully employed to study the role of various plant genes in cell-cycle control. Here, we show that TCP4 protein blocks cell cycle of budding yeast, specifically at G1 ? S transition, by regulating G1 checkpoint control pathway.
2. Materials and methods 2.1. Strains Escherichia coli strain DH5a (Promega Corp., Madison, WI) was used for bacterial manipulations of plasmids. Yeast strains used were S228c (MATa his3D1 leu2D0 met15D0 ura3D0) for growtharrest studies and SKY2596 (EY0823, SIC1::HA-kanMX6) for Sic1-stabilization studies. Yeast was cultured in YPD (1% yeast extract, 2% peptone and 2% dextrose; 2% raffinose for SKY2596) or SD (synthetic drop-out) medium with appropriate supplements at 30 °C. Yeast transformation was performed by lithium acetate method. Galactose was added at the final concentration of 2% to obtain GAL-induction.
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2.4. Immuno-blot analysis SKY2956 cells transformed with pYES2 or TCP4:VP16-M1 were grown in YP media containing 2% raffinose (YPR) and synchronized at G1. The a-factor was removed by washing cells thrice with YPR. For induction, galactose was added to the final concentration of 2%. Samples equivalent to 10 ml culture at an OD600 of 1.0 were collected at 2 h interval, cells harvested by centrifugation and re-suspended in 200 ll 20% TCA. Two hundred microliters of acidwashed glass beads were added and cells disrupted by vortexing for 5 min. Four hundred microliters of 5% TCA was added and 500 ll of the aqueous extract was transferred to a new tube. The supernatant was then spun at 3000 rpm at RT for 10 min, pellet re-suspended in 100 ll 1 Laemelli buffer, neutralized with 1 M Tris base (pH 8.0), boiled for 5 min and clarified by centrifuging for 10 min. Ten microliters of supernatant was used for immuno-blot analysis. Blots were incubated with anti-HA antibody [HA-Tag (6E2) Mouse mAb #2367, Cell Signaling Tech., Inc.] at 1:10,000 dilution with gentle shaking overnight. After 1 h incubation with secondary antibodies [HRP-conjugated anti-mouse antibody, SIGMA] at 1:10,000 dilution, protein bands were visualized using Western chemi-luminescence Kit (Pierce, Thermo Fisher Scientific). 2.5. Microarray
2.2. Plasmid construction and growth assays The coding regions of TCP4, L80H-TCP4-VP16, TCP4:VP16-M1 and TCP4:VP16-M2 were excised from pUTN104, pTEF2-L80H, pUTN064 and pUTN065, respectively, and introduced into BamHI-XhoI-digested pYES2 vector (GAL1 promoter, URA3-marked, 2 lm replicon plasmid; Invitrogen Corp., USA) to generate pUTN106-110. For spot-assay, yeast cells were grown in SD-glucose medium for 1 day, OD600 adjusted to 0.1 and diluted to various concentrations (OD600 of 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005). Aliquots (5 ll) from each were spotted onto SD-glucose or SD-galactose medium and incubated for 3–4 days at 30 °C. For growth curve, SD-glucose and SD-galactose media were inoculated with OD600 0.1 and cells were grown at 30 °C. Growth was monitored every 4 h for a day and OD600 values were recorded. 2.3. Flow cytometry Yeast cells transformed with pYES2 or TCP4:VP16-M1, grown for 16 h at 30 °C in 10 ml of SD-glucose and the culture was added to 250 ml of SD-glucose at A600 of 0.2. The a-factor was added at 8 lg/ml and culture was incubated at 30 °C for 3 h. Small aliquots were removed before and after adding a-factor for flow cytometry analysis, to test the extent of synchronization. Cells were pelleted (3000 rpm, 4 min) and washed twice with 20 ml of SD-glucose. Equal amount of re-suspended cells were added into fresh culture media of SD-glucose and SD-galactose (125 ml) and grown at 30 °C for 16 h. Samples were pelleted at 0, 4, 8 and 16 h and A600 adjusted to 0.2 in 1 PBS. 250 ll of cells was fixed by mixing with 750 ll of absolute ethanol and stored at 20 °C overnight. Fixed cells were collected by centrifugation and suspended in 1 ml of 1 PBS. Samples were sonicated (40% duty cycle, 10–12 pulses) and treated with 10 ll RNase A (10 mg/ml) and 10 ll Propidium Iodide (1 mg/ml). Staining was carried out by incubating the cells at 37 °C for 12 h in dark with gentle rotation. Flow cytometry was carried out with FL2-H detector of Becton Dickinson FACScan™ and events were analyzed with WinMDI 2.9 software (Joseph Trotter). Percentage of DNA content was calculated by dividing the number of events under each DNA peak by the total number of events under all peaks.
Total RNA was extracted from TCP4 and vector induced (galactose) samples at 0 and 4 h using Trizol (Sigma). Same RNA samples were used for RT-PCR and microarray studies. Samples were labeled with Cy-3/Cy-5 and competitively hybridized to the Agilent-yeast arrays (8 15 K) as per the manufacturer guidelines. Data analysis was carried out using GeneSpring GX version 7.3 and Microsoft Excel and normalized using GeneSpring GX feature: Per Spot and Per Chip: Intensity dependent (Lowess) normalization. Genes with >2-fold difference between the samples were considered as significantly differentially regulated genes. 3. Results and discussion 3.1. TCP4 activity arrests yeast growth We observed that expression of full-length TCP4 protein under the regulation of a strong promoter such as PTEF1 reduces growth of wild type yeast cells (Fig. 1A) [19]. To check if this growth retardation is specific to TCP4 activity, we expressed mutant forms of TCP4 and studied their effect on yeast growth. A Gly57Pro58? Leu57Leu58 double mutant [19] in the TCP domain, that abolished the DNA-binding ability of TCP4, did not retard yeast growth. Furthermore, a truncated version of TCP4 protein, that retains the DNA-binding domain but lacks the C-terminal domain, could not inhibit yeast growth either (Fig. 1A). These results show that the DNA-binding as well as transcriptional activation by TCP4 protein is required for growth inhibition of yeast cells. To further test the effect of TCP4 function on yeast growth, we used TCP4 proteins with enhanced transcriptional activity, achieved by fusing the viral protein VP16, a potent activator of transcription, with the full-length TCP4. Three such fusion proteins, TCP4:VP16M1, TCP4:VP16-M2 and TCP4:VP16-C, have been reported to enhance transcription in yeast [20]. Transformation of yeast with TCP4:VP16-C construct under PTEF1 promoter failed to yield transformants, suggesting that hyper-activation of TCP4 is lethal to yeast, possibly due to excessive growth inhibition. We, therefore, expressed TCP4-VP16 fusion proteins under galactose-inducible expression system. Growth of yeast was arrested when TCP4: VP16-M1 or TCP4:VP16-M2 proteins were induced by galactose (Fig. 1B). Such growth inhibition was not observed when wild
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Fig. 1. TCP4 activity inhibits yeast growth. (A) Growth assay of serially-diluted yeast cells expressing the indicated forms of TCP4 protein under TEF1 promoter. pYES2, empty vector used as control; TCP4, wild type TCP4 protein; GP?LL, TCP4 protein with Gly57Leu;Pro58Leu mutation; D-cter, TCP4 protein lacking the C-terminal trans-activation domain. (B) Growth assay of serially-diluted yeast cells expressing the indicated forms of TCP4 protein under GAL1 promoter grown in glucose or galactose-containing medium. pYES2, empty vector control; TCP4, full-length TCP4 protein; L80H, TCP4 protein with Leu80His mutation; TCP4DD, TCP4 protein lacking the DNA-binding domain; TCP4:VP16-M1 & TCP4:VP16-M2, TCP4 protein with in-frame VP16 insertion at 300 and 660 bp downstream to the translation start site, respectively. (C, D) Growth of yeast harboring pYES2 (}), L80H-VP16 (4) and TCP4:VP16-M1 (h) is expressed as optical density at 600 nm as a function of time. Cells were grown in liquid culture in presence of glucose (C) or galactose (D). The experiment was repeated 3 times and similar results were obtained. (E) RT-PCR analysis to show that galactose (Gal) induction increases the expression of TCP4:VP16-M1 transcript compared to when grown in glucose (Glu). Actin was used as internal control.
type TCP4 or its mutant forms [19] lacking DNA-binding or trans-activation domain were expressed under the same promoter. This demonstrated that the enhanced growth inhibition was due to increased transcriptional activity of the TCP4:VP16-M1 or TCP4:VP16-M2 proteins. The TCP4-induced growth inhibition was also observed in yeast grown in liquid medium. When expression of TCP4:VP16-M1 protein was induced by galactose, yeast growth was significantly reduced compared to vector control (Fig. 1C, D). No such growth difference was observed prior to induction in glucose-containing medium. Induction of L80H-VP16 expression reduced growth to a moderate extent, perhaps because the mutant retains residual
DNA-binding property. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis on RNA extracted from G1-synchronized yeast cells showed that the TCP4:VP16-M1 fusion transcript was 3 times up-regulated upon induction in comparison to the control cells (Fig. 1E). These results clearly establish that activation of TCP4 transcript is responsible for growth arrest in yeast cells. Thus, effect of TCP4 on repression of cell proliferation is intrinsic to the protein activity and it works in a heterologous system such as yeast. In Antirrhinum, CINCINNATA represses cell proliferation specifically in the leaf margins, where it is expressed at higher level [21]. Similar effect is also observed in yeast, where TCP4 functions in a dose-dependent manner to repress cell division. By using
Fig. 2. (A–D) Cellular morphology of haploid yeast cells upon TCP4 activation. Cells transformed with empty vector pYES2 (A & B) and TCP4:VP16-M1 (C & D) were shifted from glucose (A & C) to galactose (B & D) and observed after 4 h using light microscope. Circles in (D) highlights the ‘shmooed’ cells. (E–H) Flow cytometry studies of haploid yeast cells. X-axis represents DNA content and the two peaks along this axis correspond to 1n (G1) and 2n (G2) phases, respectively. Y-axis represents the duration (in hours) cells were induced with galactose. Control cells (E) and TCP4:VP16-M1 cells (G) in non-inducive glucose medium showed normal cell cycle progression. Upon galactose induction, control cells (F) showed normal cell cycle whereas TCP4:VP16-M1 cells (H) showed complete arrest of cell cycle at G1-phase (1n).
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Fig. 3. (A) G1 checkpoint control pathway in yeast. Major proteins involved in the pathway are shown and the corresponding genes with altered expression upon TCP4:VP16M1 induction are shown in bold. Induction of the empty vector (pYES2) was used as control for comparison. Numbers in parentheses indicate fold-difference. Down and upregulation are shown as ; and ", respectively. (B) A western blot to show that HA-Sic1 fusion protein was stabilized upon TCP4:VP16-M1 expression after 4 (S4) & 6 (S6) hours of galactose-induction. Vector-transformed cells were used as negative control (pYES2). Monoclonal antibody against HA-tag (a-HA) was used to probe the blot. S and U indicate SKY2596 cells synchronized and unsynchronized, respectively, using a-mating factor. C indicates total protein from yeast expressing untagged Sic1. Lower panel shows part of the identical silver-stained SDS–PAGE gel as loading control.
various mutant forms of TCP4 protein, we ruled out the possibility that TCP4 sequestration of some other protein by physical interaction is responsible for its effect on yeast growth. 3.2. TCP4 inhibits G1 ? S transition in yeast Since cell cycle progression in yeast is usually associated with change in cell morphology [22], we monitored the morphology of TCP4-expressing cells. Upon galactose induction, a proportion of yeast cells expressing TCP4:VP16-M1 protein showed elongated morphology compared to the cells with vector control (Fig. 2A–D). This cellular appearance in part resembles the ‘shmoo’ phenotype [23], usually observed in G1-arresrted yeast cells [24]. Thus, our results indicate that TCP4 blocks G1 ? S transition in yeast. To validate the TCP4-mediated G1-arrest, we analyzed cell cycle profile of yeast expressing TCP4 protein by flow-cytometry. Cells were synchronized at the G1 phase and induced by galactose. Synchronized cells transformed with empty vector entered into mitotic phase at 4 h and continued normal cell cycling (Fig. 2E, F). Although galactose slowed down the cell cycle progression to a small extent, the global profile of cell cycling remained unchanged. In contrast, when TCP4:VP16-M1 protein was induced, cell cycle remained arrested at G1 phase even after 16 h of induction (Fig. 2G, H). In non-inductive conditions, these cells showed normal cell cycle progression, although cell cycling rate was slightly
slower, perhaps due to basal-level expression of TCP4:VP16-M1 transcript in glucose (Fig. 1E). These results confirm that TCP4 activity blocks yeast cell cycle at G1 ? S transition. In many eukaryotic cells including yeast, G1 checkpoint is the major target of growth factors and nutritional signaling. In plants, availability of carbon source in the form of sucrose plays a major role in G1 ? S transition by controlling the expression of cyclinD [25]. CIN and TCP4 are expressed in the actively dividing leaf cells and the S-phase cells become more abundant when these genes are mutated [3,6]. It is possible that the CIN/TCP4-like TCP genes block plant cell division at the G1 ? S transitions upon induction of some differentiation signal. 3.3. Gene products involved in G1 checkpoint control are affected upon TCP4 induction To determine the basis of TCP4-induced G1-arrest, we carried out genome-wide transcript profiling of yeast cells expressing TCP4. Both TCP4:VP16-M1-expressing cells and vector-transformed control cells were synchronized at G1-phase and induced by galactose for 4 h. By using 2-fold difference as cut-off, we identified 760 transcripts that were differentially expressed in TCP4:VP16M1-expressing cells as opposed to 1400 transcripts found in control cells upon galactose induction. Fewer differentiallyexpressed genes in the TCP4:VP16-M1 cells could be an indirect
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measure of reduced cell cycling and might involve genes required for G1 ? S transition. Hence, we compared the two datasets and identified 30 genes inversely affected in TCP4:VP16-M1 and control cells after induction (Supplementary information, Table 1). Most of these genes are known to be involved in cell-cycle regulation and nearly half of them mapped to G1-checkpoint control pathway (Fig. 3A). Furthermore, 39 genes that belong to the ribosome biogenesis pathway are down-regulated in TCP4:VP16-M1 cells (p-value 7.3 1015) whereas 26 genes of the same pathway are found to be up regulated in the control dataset (p-value 2.1 102), suggestive of reduced metabolism in the former. DNA damage at G1 phase blocks accumulation of G1-cyclins (Cln)-Cdc28 complex (Fig. 3A) [26], followed by stabilization of Sic1, which maintains G1 arrest by sequestering Clb-Cdc28. DNA damage also induces another non-essential CDK, Pho85, which interacts with Pcl-type of cyclins [27,28]. The Pcls have been placed into two groups: one (such as Pcl1p, Pcl2p, etc.) postulated to function in G1 cell cycle controls and the other (Pho80p, Pcl7, etc.) in metabolic regulation (reviewed in [29,30]. Sic1 is the direct target of repression by Pcl1/Pho85 [31]. Pho80–Pho85 complex further promote G1 checkpoint by inducing Sic1 proteolysis, thus, overriding checkpoint signaling and facilitates the entry in S-phase [26]. In our microarray experiment, we found that expression of both Pcl1 and Pho85 was significantly more in control cells whereas level of Pcl5 transcript was reduced in TCP4-induced cells. In accordance to normal cycling of control cells, we found that expression of S-phase cyclins such as Clb5 was increased. Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We observed CAK1 activation in the TCP4-VP16 cells, suggesting that Cln-Cdc28 is more active in these cells. The transcript of Cln3, a promoter of G1 ? S transition, was decreased 2-fold in the TCP4-induced cells, suggestive of defective G1-phase progression. Since Sic1 is the direct target of Pcl1–Pho85 and Cln-Cdc28 mediated degradation, we postulated that Sic1 stabilization in TCP4-expressing cells contributes to the G1 arrest. An immunoblotting experiment using anti-HA antibody as probe showed that significantly higher level of HA-Sic1 fusion protein accumulated in TCP4-VP16 expressing cells compared to the control cells (Fig. 3B). Thus, TCP4 expression in yeast arrests the cell cycle at G1 ? S transition by preventing the degradation of CDK inhibitor, Sic1. Results summarized in Fig. 3 suggest that Pho80–Pho85 complex has decreased activity in TCP4 expressing yeast cells compared to the control cells. Reduced degradation of Sic1 protein in TCP4 expressing cells confirms our prediction. Similar stabilization of Sic1 was also observed in pho85D mutant cells that are arrested at G1 with elongated buds [31]. Decrease in Sic1 phosphorylation due to low levels of Pho80–Pho85 kinase is perhaps the major contributor to its stabilization. Many plant CKIs have been identified in the last few years that are implicated in cell cycle control during cell differentiation [32,33]. TCP4-mediated stabilization of CKI might also be employed by plants to repress cell division and to promote cell differentiation during the growth of leaf. Sic1 homologues are, however, not present in plants [32], but other CKIs possibly have replaced Sic1 function. Our results demonstrate that TCP4 is involved in direct repression of cell division at G1 ? S transition. Whether such growth arrest is mediated through the stabilization of CKIs in plants also, is yet to be determined. Acknowledgments We thank Dr. Steven Kron, University of Chicago for the yeast HA-Sic1 strain. PA was supported by a fellowship from Council for Scientific and Industrial Research, Govt. of India. PPS and UN acknowledge the financial support from Department of Biotechnology, Govt. of India.
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The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 ? S transition Pooja Aggarwal 1, Bhavna Padmanabhan, Abhay Bhat, Kavitha Sarvepalli, Parag P. Sadhale, Utpal Nath ⇑ Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e
i n f o
Article history: Received 21 May 2011 Available online 30 May 2011 Keywords: TCP transcription factor Arabidopsis Cell cycle Budding yeast G1 ? S transition
a b s t r a c t The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 ? S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 ? S arrest is discussed. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction TCP proteins are plant-specific transcription factors that control diverse functions in plants [1–6]. They share a conserved DNAbinding domain, known as TCP domain, that folds into basic helix–loop–helix structure. TCP proteins are classified into two groups; class I and II [7,8]. Several studies indicate that class I TCP proteins promote cell division whereas class II proteins repress organ growth by inhibiting cell proliferation [9]. For example, class I TCP proteins PCF1 and PCF2 in rice bind to the PCNA promoter [10] and TCP20 in Arabidopsis interacts with the cis-element of cyclin B [11]. However, a direct link between class II TCP proteins and cell cycle genes has not been demonstrated. CINCINNATA (CIN) in Antirrhinum and TCP4 in Arabidopsis are homologous class II TCP proteins that control leaf morphogenesis by repressing cell division [3,4]. Although it has been postulated that CIN alters the transcriptional status of cell cycle genes [3], its exact mechanism is poorly understood. When multiple CIN-like TCP genes were mutated in Arabidopsis, the mitotic index in developing leaves was not significantly altered [6], suggesting an indirect role for CIN-like TCP proteins in controlling cell cycle. To investigate the effect of TCP proteins on cell cycle more objectively and in greater detail, we have studied their effect on cell division in budding yeast, an organism that lacks TCP-like genes.
⇑ Corresponding author. E-mail address: [email protected] (U. Nath). Present address: Temasek Life Sciences Laboratory Limited, 1 Research Link, National University of Singapore, Singapore 117604, Singapore. 1
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.132
Cell cycle is remarkably conserved in yeast, animals, and plants and is controlled by hetero-dimeric CDK (cyclin-dependent kinases)/cyclin complexes [12]. Three major checkpoints control the execution of cell cycle: G1 ? S, G2 ? M and metaphase ? anaphase transitions. Budding yeast has a single CDK, Cdc28, that regulates progression through all phases of cell cycle in association with different cyclin partners, such as Cln1–3 (G1 cyclin), Clb5–6 (S cyclin) and Clb1–4 (M cyclin) [13]. Arabidopsis has several classes of CDKs (CDKA-F) and more than 49 cyclins [14]. A & B classes of CDKs are the two major drivers of plant cell cycle. CDKAs share homology with yeast and mammalian CDKs and control both G1–S and G2–M transitions. CDKBs are specific to plants and are restricted to G2–M phase. Among several classes of cyclins identified in plants, CYCA, CYCB and CYCD constitute three major groups. CYCD, in association with CDKA, regulates G1 ? S transition. Activity of CDK–cyclin complexes is further regulated by proteins such as CDK-activating enzymes (CAK) and CDK inhibitory proteins (CKIs) [15,16]. Regulation of cell cycle by CKIs is conserved in yeast, animals and plants. These proteins act as the regulators of G1 ? S checkpoint in response to environmental and developmental cues and thus orchestrate eukaryotic cell proliferation and differentiation [16]. In budding yeast, the major CKI, Sic1, prevents premature S phase initiation until Cdc28/Cln1–2 levels are sufficient to complete G1 phase successfully [17]. Direct effect of various genes on the plant cell-cycle regulation is more difficult to study mainly due to the complexity of the core cell cycle components in plants. Conservation of the basic tools of the cell cycle machinery among plants and yeast has, however, enabled the isolation of many plant CDKs and cyclins by comparative
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genomics. For instance, genetic complementation of G1 cyclindeficient yeast mutant cells has led to the cloning of cyclin D in plants [18], suggesting that yeast can be successfully employed to study the role of various plant genes in cell-cycle control. Here, we show that TCP4 protein blocks cell cycle of budding yeast, specifically at G1 ? S transition, by regulating G1 checkpoint control pathway.
2. Materials and methods 2.1. Strains Escherichia coli strain DH5a (Promega Corp., Madison, WI) was used for bacterial manipulations of plasmids. Yeast strains used were S228c (MATa his3D1 leu2D0 met15D0 ura3D0) for growtharrest studies and SKY2596 (EY0823, SIC1::HA-kanMX6) for Sic1-stabilization studies. Yeast was cultured in YPD (1% yeast extract, 2% peptone and 2% dextrose; 2% raffinose for SKY2596) or SD (synthetic drop-out) medium with appropriate supplements at 30 °C. Yeast transformation was performed by lithium acetate method. Galactose was added at the final concentration of 2% to obtain GAL-induction.
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2.4. Immuno-blot analysis SKY2956 cells transformed with pYES2 or TCP4:VP16-M1 were grown in YP media containing 2% raffinose (YPR) and synchronized at G1. The a-factor was removed by washing cells thrice with YPR. For induction, galactose was added to the final concentration of 2%. Samples equivalent to 10 ml culture at an OD600 of 1.0 were collected at 2 h interval, cells harvested by centrifugation and re-suspended in 200 ll 20% TCA. Two hundred microliters of acidwashed glass beads were added and cells disrupted by vortexing for 5 min. Four hundred microliters of 5% TCA was added and 500 ll of the aqueous extract was transferred to a new tube. The supernatant was then spun at 3000 rpm at RT for 10 min, pellet re-suspended in 100 ll 1 Laemelli buffer, neutralized with 1 M Tris base (pH 8.0), boiled for 5 min and clarified by centrifuging for 10 min. Ten microliters of supernatant was used for immuno-blot analysis. Blots were incubated with anti-HA antibody [HA-Tag (6E2) Mouse mAb #2367, Cell Signaling Tech., Inc.] at 1:10,000 dilution with gentle shaking overnight. After 1 h incubation with secondary antibodies [HRP-conjugated anti-mouse antibody, SIGMA] at 1:10,000 dilution, protein bands were visualized using Western chemi-luminescence Kit (Pierce, Thermo Fisher Scientific). 2.5. Microarray
2.2. Plasmid construction and growth assays The coding regions of TCP4, L80H-TCP4-VP16, TCP4:VP16-M1 and TCP4:VP16-M2 were excised from pUTN104, pTEF2-L80H, pUTN064 and pUTN065, respectively, and introduced into BamHI-XhoI-digested pYES2 vector (GAL1 promoter, URA3-marked, 2 lm replicon plasmid; Invitrogen Corp., USA) to generate pUTN106-110. For spot-assay, yeast cells were grown in SD-glucose medium for 1 day, OD600 adjusted to 0.1 and diluted to various concentrations (OD600 of 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005). Aliquots (5 ll) from each were spotted onto SD-glucose or SD-galactose medium and incubated for 3–4 days at 30 °C. For growth curve, SD-glucose and SD-galactose media were inoculated with OD600 0.1 and cells were grown at 30 °C. Growth was monitored every 4 h for a day and OD600 values were recorded. 2.3. Flow cytometry Yeast cells transformed with pYES2 or TCP4:VP16-M1, grown for 16 h at 30 °C in 10 ml of SD-glucose and the culture was added to 250 ml of SD-glucose at A600 of 0.2. The a-factor was added at 8 lg/ml and culture was incubated at 30 °C for 3 h. Small aliquots were removed before and after adding a-factor for flow cytometry analysis, to test the extent of synchronization. Cells were pelleted (3000 rpm, 4 min) and washed twice with 20 ml of SD-glucose. Equal amount of re-suspended cells were added into fresh culture media of SD-glucose and SD-galactose (125 ml) and grown at 30 °C for 16 h. Samples were pelleted at 0, 4, 8 and 16 h and A600 adjusted to 0.2 in 1 PBS. 250 ll of cells was fixed by mixing with 750 ll of absolute ethanol and stored at 20 °C overnight. Fixed cells were collected by centrifugation and suspended in 1 ml of 1 PBS. Samples were sonicated (40% duty cycle, 10–12 pulses) and treated with 10 ll RNase A (10 mg/ml) and 10 ll Propidium Iodide (1 mg/ml). Staining was carried out by incubating the cells at 37 °C for 12 h in dark with gentle rotation. Flow cytometry was carried out with FL2-H detector of Becton Dickinson FACScan™ and events were analyzed with WinMDI 2.9 software (Joseph Trotter). Percentage of DNA content was calculated by dividing the number of events under each DNA peak by the total number of events under all peaks.
Total RNA was extracted from TCP4 and vector induced (galactose) samples at 0 and 4 h using Trizol (Sigma). Same RNA samples were used for RT-PCR and microarray studies. Samples were labeled with Cy-3/Cy-5 and competitively hybridized to the Agilent-yeast arrays (8 15 K) as per the manufacturer guidelines. Data analysis was carried out using GeneSpring GX version 7.3 and Microsoft Excel and normalized using GeneSpring GX feature: Per Spot and Per Chip: Intensity dependent (Lowess) normalization. Genes with >2-fold difference between the samples were considered as significantly differentially regulated genes. 3. Results and discussion 3.1. TCP4 activity arrests yeast growth We observed that expression of full-length TCP4 protein under the regulation of a strong promoter such as PTEF1 reduces growth of wild type yeast cells (Fig. 1A) [19]. To check if this growth retardation is specific to TCP4 activity, we expressed mutant forms of TCP4 and studied their effect on yeast growth. A Gly57Pro58? Leu57Leu58 double mutant [19] in the TCP domain, that abolished the DNA-binding ability of TCP4, did not retard yeast growth. Furthermore, a truncated version of TCP4 protein, that retains the DNA-binding domain but lacks the C-terminal domain, could not inhibit yeast growth either (Fig. 1A). These results show that the DNA-binding as well as transcriptional activation by TCP4 protein is required for growth inhibition of yeast cells. To further test the effect of TCP4 function on yeast growth, we used TCP4 proteins with enhanced transcriptional activity, achieved by fusing the viral protein VP16, a potent activator of transcription, with the full-length TCP4. Three such fusion proteins, TCP4:VP16M1, TCP4:VP16-M2 and TCP4:VP16-C, have been reported to enhance transcription in yeast [20]. Transformation of yeast with TCP4:VP16-C construct under PTEF1 promoter failed to yield transformants, suggesting that hyper-activation of TCP4 is lethal to yeast, possibly due to excessive growth inhibition. We, therefore, expressed TCP4-VP16 fusion proteins under galactose-inducible expression system. Growth of yeast was arrested when TCP4: VP16-M1 or TCP4:VP16-M2 proteins were induced by galactose (Fig. 1B). Such growth inhibition was not observed when wild
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Fig. 1. TCP4 activity inhibits yeast growth. (A) Growth assay of serially-diluted yeast cells expressing the indicated forms of TCP4 protein under TEF1 promoter. pYES2, empty vector used as control; TCP4, wild type TCP4 protein; GP?LL, TCP4 protein with Gly57Leu;Pro58Leu mutation; D-cter, TCP4 protein lacking the C-terminal trans-activation domain. (B) Growth assay of serially-diluted yeast cells expressing the indicated forms of TCP4 protein under GAL1 promoter grown in glucose or galactose-containing medium. pYES2, empty vector control; TCP4, full-length TCP4 protein; L80H, TCP4 protein with Leu80His mutation; TCP4DD, TCP4 protein lacking the DNA-binding domain; TCP4:VP16-M1 & TCP4:VP16-M2, TCP4 protein with in-frame VP16 insertion at 300 and 660 bp downstream to the translation start site, respectively. (C, D) Growth of yeast harboring pYES2 (}), L80H-VP16 (4) and TCP4:VP16-M1 (h) is expressed as optical density at 600 nm as a function of time. Cells were grown in liquid culture in presence of glucose (C) or galactose (D). The experiment was repeated 3 times and similar results were obtained. (E) RT-PCR analysis to show that galactose (Gal) induction increases the expression of TCP4:VP16-M1 transcript compared to when grown in glucose (Glu). Actin was used as internal control.
type TCP4 or its mutant forms [19] lacking DNA-binding or trans-activation domain were expressed under the same promoter. This demonstrated that the enhanced growth inhibition was due to increased transcriptional activity of the TCP4:VP16-M1 or TCP4:VP16-M2 proteins. The TCP4-induced growth inhibition was also observed in yeast grown in liquid medium. When expression of TCP4:VP16-M1 protein was induced by galactose, yeast growth was significantly reduced compared to vector control (Fig. 1C, D). No such growth difference was observed prior to induction in glucose-containing medium. Induction of L80H-VP16 expression reduced growth to a moderate extent, perhaps because the mutant retains residual
DNA-binding property. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis on RNA extracted from G1-synchronized yeast cells showed that the TCP4:VP16-M1 fusion transcript was 3 times up-regulated upon induction in comparison to the control cells (Fig. 1E). These results clearly establish that activation of TCP4 transcript is responsible for growth arrest in yeast cells. Thus, effect of TCP4 on repression of cell proliferation is intrinsic to the protein activity and it works in a heterologous system such as yeast. In Antirrhinum, CINCINNATA represses cell proliferation specifically in the leaf margins, where it is expressed at higher level [21]. Similar effect is also observed in yeast, where TCP4 functions in a dose-dependent manner to repress cell division. By using
Fig. 2. (A–D) Cellular morphology of haploid yeast cells upon TCP4 activation. Cells transformed with empty vector pYES2 (A & B) and TCP4:VP16-M1 (C & D) were shifted from glucose (A & C) to galactose (B & D) and observed after 4 h using light microscope. Circles in (D) highlights the ‘shmooed’ cells. (E–H) Flow cytometry studies of haploid yeast cells. X-axis represents DNA content and the two peaks along this axis correspond to 1n (G1) and 2n (G2) phases, respectively. Y-axis represents the duration (in hours) cells were induced with galactose. Control cells (E) and TCP4:VP16-M1 cells (G) in non-inducive glucose medium showed normal cell cycle progression. Upon galactose induction, control cells (F) showed normal cell cycle whereas TCP4:VP16-M1 cells (H) showed complete arrest of cell cycle at G1-phase (1n).
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Fig. 3. (A) G1 checkpoint control pathway in yeast. Major proteins involved in the pathway are shown and the corresponding genes with altered expression upon TCP4:VP16M1 induction are shown in bold. Induction of the empty vector (pYES2) was used as control for comparison. Numbers in parentheses indicate fold-difference. Down and upregulation are shown as ; and ", respectively. (B) A western blot to show that HA-Sic1 fusion protein was stabilized upon TCP4:VP16-M1 expression after 4 (S4) & 6 (S6) hours of galactose-induction. Vector-transformed cells were used as negative control (pYES2). Monoclonal antibody against HA-tag (a-HA) was used to probe the blot. S and U indicate SKY2596 cells synchronized and unsynchronized, respectively, using a-mating factor. C indicates total protein from yeast expressing untagged Sic1. Lower panel shows part of the identical silver-stained SDS–PAGE gel as loading control.
various mutant forms of TCP4 protein, we ruled out the possibility that TCP4 sequestration of some other protein by physical interaction is responsible for its effect on yeast growth. 3.2. TCP4 inhibits G1 ? S transition in yeast Since cell cycle progression in yeast is usually associated with change in cell morphology [22], we monitored the morphology of TCP4-expressing cells. Upon galactose induction, a proportion of yeast cells expressing TCP4:VP16-M1 protein showed elongated morphology compared to the cells with vector control (Fig. 2A–D). This cellular appearance in part resembles the ‘shmoo’ phenotype [23], usually observed in G1-arresrted yeast cells [24]. Thus, our results indicate that TCP4 blocks G1 ? S transition in yeast. To validate the TCP4-mediated G1-arrest, we analyzed cell cycle profile of yeast expressing TCP4 protein by flow-cytometry. Cells were synchronized at the G1 phase and induced by galactose. Synchronized cells transformed with empty vector entered into mitotic phase at 4 h and continued normal cell cycling (Fig. 2E, F). Although galactose slowed down the cell cycle progression to a small extent, the global profile of cell cycling remained unchanged. In contrast, when TCP4:VP16-M1 protein was induced, cell cycle remained arrested at G1 phase even after 16 h of induction (Fig. 2G, H). In non-inductive conditions, these cells showed normal cell cycle progression, although cell cycling rate was slightly
slower, perhaps due to basal-level expression of TCP4:VP16-M1 transcript in glucose (Fig. 1E). These results confirm that TCP4 activity blocks yeast cell cycle at G1 ? S transition. In many eukaryotic cells including yeast, G1 checkpoint is the major target of growth factors and nutritional signaling. In plants, availability of carbon source in the form of sucrose plays a major role in G1 ? S transition by controlling the expression of cyclinD [25]. CIN and TCP4 are expressed in the actively dividing leaf cells and the S-phase cells become more abundant when these genes are mutated [3,6]. It is possible that the CIN/TCP4-like TCP genes block plant cell division at the G1 ? S transitions upon induction of some differentiation signal. 3.3. Gene products involved in G1 checkpoint control are affected upon TCP4 induction To determine the basis of TCP4-induced G1-arrest, we carried out genome-wide transcript profiling of yeast cells expressing TCP4. Both TCP4:VP16-M1-expressing cells and vector-transformed control cells were synchronized at G1-phase and induced by galactose for 4 h. By using 2-fold difference as cut-off, we identified 760 transcripts that were differentially expressed in TCP4:VP16M1-expressing cells as opposed to 1400 transcripts found in control cells upon galactose induction. Fewer differentiallyexpressed genes in the TCP4:VP16-M1 cells could be an indirect
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measure of reduced cell cycling and might involve genes required for G1 ? S transition. Hence, we compared the two datasets and identified 30 genes inversely affected in TCP4:VP16-M1 and control cells after induction (Supplementary information, Table 1). Most of these genes are known to be involved in cell-cycle regulation and nearly half of them mapped to G1-checkpoint control pathway (Fig. 3A). Furthermore, 39 genes that belong to the ribosome biogenesis pathway are down-regulated in TCP4:VP16-M1 cells (p-value 7.3 1015) whereas 26 genes of the same pathway are found to be up regulated in the control dataset (p-value 2.1 102), suggestive of reduced metabolism in the former. DNA damage at G1 phase blocks accumulation of G1-cyclins (Cln)-Cdc28 complex (Fig. 3A) [26], followed by stabilization of Sic1, which maintains G1 arrest by sequestering Clb-Cdc28. DNA damage also induces another non-essential CDK, Pho85, which interacts with Pcl-type of cyclins [27,28]. The Pcls have been placed into two groups: one (such as Pcl1p, Pcl2p, etc.) postulated to function in G1 cell cycle controls and the other (Pho80p, Pcl7, etc.) in metabolic regulation (reviewed in [29,30]. Sic1 is the direct target of repression by Pcl1/Pho85 [31]. Pho80–Pho85 complex further promote G1 checkpoint by inducing Sic1 proteolysis, thus, overriding checkpoint signaling and facilitates the entry in S-phase [26]. In our microarray experiment, we found that expression of both Pcl1 and Pho85 was significantly more in control cells whereas level of Pcl5 transcript was reduced in TCP4-induced cells. In accordance to normal cycling of control cells, we found that expression of S-phase cyclins such as Clb5 was increased. Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We observed CAK1 activation in the TCP4-VP16 cells, suggesting that Cln-Cdc28 is more active in these cells. The transcript of Cln3, a promoter of G1 ? S transition, was decreased 2-fold in the TCP4-induced cells, suggestive of defective G1-phase progression. Since Sic1 is the direct target of Pcl1–Pho85 and Cln-Cdc28 mediated degradation, we postulated that Sic1 stabilization in TCP4-expressing cells contributes to the G1 arrest. An immunoblotting experiment using anti-HA antibody as probe showed that significantly higher level of HA-Sic1 fusion protein accumulated in TCP4-VP16 expressing cells compared to the control cells (Fig. 3B). Thus, TCP4 expression in yeast arrests the cell cycle at G1 ? S transition by preventing the degradation of CDK inhibitor, Sic1. Results summarized in Fig. 3 suggest that Pho80–Pho85 complex has decreased activity in TCP4 expressing yeast cells compared to the control cells. Reduced degradation of Sic1 protein in TCP4 expressing cells confirms our prediction. Similar stabilization of Sic1 was also observed in pho85D mutant cells that are arrested at G1 with elongated buds [31]. Decrease in Sic1 phosphorylation due to low levels of Pho80–Pho85 kinase is perhaps the major contributor to its stabilization. Many plant CKIs have been identified in the last few years that are implicated in cell cycle control during cell differentiation [32,33]. TCP4-mediated stabilization of CKI might also be employed by plants to repress cell division and to promote cell differentiation during the growth of leaf. Sic1 homologues are, however, not present in plants [32], but other CKIs possibly have replaced Sic1 function. Our results demonstrate that TCP4 is involved in direct repression of cell division at G1 ? S transition. Whether such growth arrest is mediated through the stabilization of CKIs in plants also, is yet to be determined. Acknowledgments We thank Dr. Steven Kron, University of Chicago for the yeast HA-Sic1 strain. PA was supported by a fellowship from Council for Scientific and Industrial Research, Govt. of India. PPS and UN acknowledge the financial support from Department of Biotechnology, Govt. of India.
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