Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

Authors

  • Manas K. Chattopadhyay,

    Corresponding author
    1. Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA
    • NIDDK, National Institutes of Health, 8 Center Drive, Building 8, Room 219, Bethesda, MD 20892-0830, USA.
    Search for more papers by this author
  • Weiping Chen,

    1. Microarray Core Facility, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA
    Search for more papers by this author
  • George Poy,

    1. Microarray Core Facility, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA
    Search for more papers by this author
  • Margaret Cam,

    1. Microarray Core Facility, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA
    Search for more papers by this author
  • David Stiles,

    1. Microarray Core Facility, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA
    Search for more papers by this author
  • Herbert Tabor

    1. Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA
    Search for more papers by this author

Abstract

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well-established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10−5M) or spermine (10−5M) compared to a control culture containing 10−8M spermidine. 247 genes were upregulated > two-fold and 11 genes were upregulated >10-fold after spermidine addition. Functional categorization of the genes showed induction of transport-related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two-fold, and six genes were downregulated > eight-fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.

Introduction

Polyamines are found in essentially all cellular organisms and intracellular concentrations are controlled at various steps (Tabor and Tabor, 1984; Pegg, 1986; Cohen, 1998; Igarashi and Kashiwagi, 2000). The essential role of polyamines has been shown by using various inhibitors of polyamine biosynthesis or by using mutants in the biosynthetic pathways and they have been shown to have a variety of phenotypic effects (Mamont et al., 1981; Diala et al., 1980; Tabor and Tabor, 1984; Pitkin and Davis, 1990; Cohen, 1998). In addition to the ionic interaction of polyamines with negatively charged macromolecules (such as nucleic acids, membranes and ion channels), polyamines can be covalently incorporated into proteins. One of the important functions of spermidine is its role as a substrate for the hypusine modification of a 17 000–18 000 Da protein, known as eukaryotic initiation factor 5A (eIF5A), which is a ubiquitous initiation factor in eukaryotic cells (Park et al., 1981; Park, 2006). In our previous studies we have constructed mutants of S. cerevisiae where different steps in the biosynthetic pathway for polyamines have been deleted, and have shown that spermidine is essential for the growth of these auxotrophs (Balasundaram et al., 1991, 1993, 1999; Hamasaki-Katagiri et al., 1997; Chattopadhyay et al., 2002, 2006). More recently we have found that very low concentrations of spermidine (10−8M) are sufficient to support near-normal growth and hypusine formation of these auxotrophs of S. cerevisiae, even though under these conditions the cells contain only about 1/1000 of the internal spermidine concentration found in a wild-type strain (Chattopadhyay et al., 2008). In the current study we have taken advantage of global gene expression analysis using microarrays to address the question of why wild-type cells normally contain concentrations of spermidine that are so much higher than is required for the growth of a spermidine auxotroph, and what are the molecular targets or metabolic pathways affected by excess polyamines. We have compared the changes in gene expressions when a higher concentration of either spermidine or spermine was added to cultures growing at a near-normal growth rate with 10−8M spermidine. We have purposely not made this comparison between cells that were not growing because they were completely deprived of spermidine and cells growing normally after spermidine supplementation, since such a comparison would obviously be complicated by the profound changes in gene expression expected in growing vs. non-growing cells.

Materials and methods

Growth condition

For all the experiments in this paper, Y549 (Matα met15 leu2 ura3 spe3Δ::URA3 fms1Δ::KANMX (Chattopadhyay et al., 2003) strain was used and all cultures were grown at 30 °C with shaking in air. As described earlier, this strain cannot synthesize spermidine due to lack of spermidine synthase (SPE3; Hamasaki-Katagiri et al., 1997) and cannot oxidize spermine to spermidine due to deletion of FMS1, the gene encoding yeast spermine oxidase (White et al., 2001; Chattopadhyay et al., 2003). Yeast cultures were grown routinely in plates containing yeast extract, peptone, dextrose and agar (YPAD). To deplete intracellular amines carried over from YPAD plates, the cultures were inoculated into SD medium (0.67% yeast nitrogen base, required amino acid supplements, 2% dextrose) supplemented with 10−8M spermidine and grown for >24 h with several dilutions. The culture was then diluted 1 : 2000-fold into SD medium containing 10−8M spermidine, divided into three parts and incubated overnight. When the cultures reached OD600 = 0.1, in one part 10−5M spermidine was added; in another part 10−5M purified spermine (purified by chromatography on a Dowex 50 column; Chattopadhyay et al., 2003) was added; the third part was kept as a control without any addition. These three cultures were then grown for another 6 h at 30 °C and harvested for RNA isolation. Intracellular concentrations of polyamines after incubation were determined as previously described (Chattopadhyay et al., 2008).

Isolation of RNA

Total RNA was isolated according to the protocol described in the RNeasy mini kit (Qiagen, Germantown, MD, USA). 107–108 cells from the yeast cultures, grown and harvested as described above, were washed with 1.2 M sorbitol solution (1.2 M sorbitol, 0.1% β-mercaptoethanol, 0.1 M EDTA) and resuspended in the same solution containing 50 U lyticase (Sigma) per 107 cells. The cell suspensions were incubated for 10 min at 30 °C and the resulting spheroplasts were immediately processed for RNA isolation. The quantity and quality of RNA were evaluated by OD260/OD280 assays and by RNA capillary electrophoresis (Agilent Biotechnologies).

Microarray, statistics and bioinformatics

Total RNAs were reverse-transcribed by Superscript II (Invitrogen). Oligo (dT) linked to T7 RNA polymerase promoter sequence was used to prime cDNA synthesis. After second strand synthesis, biotinylated cRNA was made by in vitro transcription using biotinylated UTP and CTP (Bioarray high-yield RNA transcript labelling kit, ENZO Diagnostics) and purified with RNeasy columns (Qiagen). The biotinylated cRNA was heated in a buffer containing100 mM potassium acetate, 30 mM magnesium acetate, 40 mM Tris–acetate, pH 8.0, to produce 35–200 base fragments. Affymetrix (Santa Clara, CA) GeneChip S-98 arrays (∼7000 genes) (n = 5 for control and spermidine-treated samples and three for spermine-treated samples) were hybridized for 16 h at 45 °C with 15 µg fragmented cRNA. Arrays were stained with streptavidin–phycoerythrin conjugate (Molecular Probes) and visualized with a gene array scanner (Agilent, Palo Alto, CA). Probe profiler software version 3.4 was used to convert hybridization intensity data into quantitative estimates of gene expression; gene probes that were not expressed in any of the samples were considered absent and were not included for further analyses. The microarray signals were analysed using the Affymetrix RMA algorithm. Comparisons of expression analyses were performed using Affimetrix MAS version 5.0 software according to the manufacturer's method. Up- and downregulated genes were selected based on p values of < 0.05 and fold change >+2 or −2, as assessed by two-way ANOVA using Partek Pro software (Partek, St. Charles, MO, USA). Hypothetical genes and unknown ORFs were not considered in the current analyses. The complete microarray data can be obtained from (GEO Accession No. GSE15269). Enrichment in specific pathways were determined by combined annotations taken from the Saccharomyces genome (http://db.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl), Affymetrix NetAffx and gene ontology consortium (http://www.geneontology.org) databases. Significance of enrichment was assessed using hypergeometric distribution.

Real-time PCR analysis

For validation of the microarray results, five genes from the upregulated gene list were selected for real-time PCR analyses. Cultures were grown as above with 10−8M spermidine (control) or with 10−5M spermidine, and cells were harvested after 1, 2, 4 and 6 h. Each RT–PCR assay was repeated using three biological replicates and each analysis consisted of three technical replicates. Before PCR, each total RNA was processed with RNase-free DNase (Qiagen). RNA was reverse transcribed by Superscript II (Invitrogen). The primers were designed using Applied Biosystems (Foster City, CA, USA) Primer Express design software. Primers and fluorescence resonance energy transfer probes were purchased from Applied Biosystems. The RT–PCR reagents, including the 18S rRNA, assay plates and 7900 HT Fast Real-time PCR system were obtained from Applied Biosystems. Relative quantitation of transcript signals was performed by using 5-log10 standard curves with 18 S rRNA as the normalizer for each assay. Fold change was calculated by the ΔCt method (Livak and Schmittgen, 2001). Statistical significance for the calculated fold changes was set with an α value of 0.05 and the p values were then calculated for the real-time PCR results.

Results

Global gene expression in response to spermidine addition

We have applied microarray analysis to study the global gene expression profile of a logarithmically growing yeast polyamine auxotroph (spe3Δ fms1Δ) supplemented with spermidine. Our results showed that a considerable number of genes were responsive to the addition of 10−5M spermidine to a spermidine auxotroph growing in 10−8M spermidine. The changes in gene expression were measured 6 h after the higher concentration of spermidine was added to the cultures. As shown in Figure 1, there was little difference in growth rate resulting from these differences in spermidine concentration, and thus the effects found were not due to marked growth changes.

Figure 1.

Effect of the addition of 10−5M spermidine or 10−5M spermine on the growth of a yeast auxotroph growing in minimum spermidine (10−8M). Y549 culture was grown in 10−8M spermidine, as mentioned in the text, and the culture was diluted into three parts and grown overnight to OD600 = 0.1; to one part spermidine or spermine at 10−5M concentration was added and the third culture was kept as a control without further addition and incubated for 6 h. Optical density at 600 nm was noted at different time points, as mentioned in Materials and methods

A comparison of the effects of spermidine and spermine treatment is shown in Figure 2A. It is evident from the volcanic graph, that spermidine treatment has a more pronounced effect than spermine treatment. After the addition of 10−5M spermidine, transcripts of 247 genes (3.5%, expressed per total number of genes in S. cerevisiae) were upregulated > two-fold. 92 genes (1.3%) were upregulated > three-fold, 33 genes were upregulated > five-fold and 11 genes were upregulated > 10-fold. 268 genes (4.3%) were downregulated > two-fold, 50 genes (0.7%) were downregulated > four-fold and six genes were downregulated > eight-fold. To facilitate data analysis, the genes were grouped into functional categories based on the Saccharomyces genome database, Affymetrix gene ID and gene ontology (GO). Top GO categories of upregulated genes are shown in Figure 2B. Most significant categories include sulphur amino acid metabolism (including methionine and sirohaem biosynthesis), arginine biosynthesis, biotin biosynthesis, lysine and NAD biosynthesis.

Figure 2.

Changes in gene expression after addition of spermidine or spermine. (A) Volcanic plots depicting p values obtained from a two-way ANOVA on the y axis and fold change on the x axis of either spermidine (SPD, left panel) or spermine (SPM, right panel)-treated cells compared to control. (B) Top five enriched gene ontology categories (p < 0.001) showing the major metabolic pathways induced by spermidine. The graph shows the percentage of genes affected out of a total within each functional category

Real-time PCR analysis using specific probes for five of the induced genes (MET2, STR3, BIO5, SEO1 and PHO84) are shown in Figure 3. A comparison of real-time PCR and microarray data is shown in Figure 3A. Both measurements were consistent and showed induction of gene expression after spermidine treatment, although the fold increase differed in these two different measurements. A time course after the addition of 10−5M spermidine showed that induction of gene expression occurred only after 2 h of spermidine treatment (Figure 3B, C), even though the internal concentration of spermidine was elevated in less than 30 min (data not shown).

Figure 3.

Real-time PCR analyses of the several spermidine-induced genes. (A) Comparison of microarray data and real-time PCR analyses of five spermidine-induced genes 6 h after spermidine treatment. (B, C) Time course of gene expression after spermidine treatment. Genes selected for validation were BIO5 (7-keto 8-aminopelargonic acid transporter), PHO84 (inorganic phosphate transporter), CTR3 (high affinity copper transporter), MET2 (L-homoserine-O-acetyl transferase) and SEO1 (allantoate transporter subfamily)

Upregulation of genes involved in methionine and sulphur amino acid metabolism

As shown in Figure 4 and Table 1, most of the genes involved in sulphur metabolism and all of the genes in the pathway from the uptake of sulphate to homocysteine and methionine synthesis (Thomas and Surdin-Kerjan, 1997) were induced by spermidine treatment. Several genes involved in the synthesis in sirohaem (a molecule that is required for a functional sulphite reductase encoded by MET10), such as MET1, MET8 and MET10 were induced. In addition, genes encoding transporters of methionine (MUP1, MUP3), S-adenosylmethionine (SAM3) and methylmethionine (MMP1) (Isnard et al., 1996) showed enhanced expression.

Figure 4.

Effect of spermidine addition on induction of genes involved in yeast sulphate- and sulphur-containing amino acid metabolism. Genes upregulated in the biosynthesis, assimilation and transport of sulphur and sulphur-containing amino acids; fold inductions are shown in parentheses. The pathway is based on gene annotations by Saccharomyces Genome Database. APS, adenosine phosphosulfate; PAPS, phosphoadenosine phosphosulphate

Table 1. Spermidine-induced sulphur or methionine metabolism related genes*
Gene namesAnnoted functionsFold induced
  • *

    Data are average of five independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

MET2L-Homoserine-O-acetyl transferase, methionine biosynthesis16.6
STR3Sulphur transfer, cystathione β-lyase activity, methionine biosynthesis23.3
MET 14Adenyl sulphate kinase, sulphate assimilation pathway; cell12.5
MET3ATP-sulphurylase, sulphate activation, sulphate to sulphide, methionine metabolism10.3
MET10Sulphite reductase, sulphite to sulphide7.1
MHT1S-Methylmethionine homocysteine methyltransferase, sulphur amino acid metabolism7.3
MXR1Protein–methionine–sulphoxide-reductase activity, response to oxidative stress8.3
MET22Bisphosphate-3-nucleotidase, salt tolerance, methionine biosynthesis8.3
MET 28Sulphur metabolism, transcriptional activator5
MET8Bifunctional dehydrogenase and ferrochelatase, sirohaem biosynthesis, sulphate assimilation5.8
MET32Zinc finger DNA binding transcriptional regulator protein, sulphur amino acid metabolism5
MET16Phosphoadenylyl-sulphate reductase (thioredoxin) activity, methionine metabolism5.2
MET1S-Adenosyl-L-methionine uroporphyrinogen transmethylase, sulphate assimilation: sirohaem biosynthesis4.7
MET13Methyl tetrahydrofolate reductase, sulphur amino acid biosynthesis3.2
MET6Cobalamine-independent methionine synthase, methionine biosynthesis2.7
SUL1Sulphate transporter activity, control the endogenous concentration of activated sulphur intermediates7.8
SUL2Sulphate transporter activity, control the endogenous concentration of activated sulphur intermediates4.6
SAM3S-Adenosylmethionine permease, required for utilization of S-adenosylmethionine as a sulphur source4
BDS1Alkyl sulphatase activity, bacterially-derived sulphatase2.8
ECM17Sulphite reductase β-subunit, involved in amino acid biosynthesis, repressed by methionine9.4
MMP1High affinity S-methylmethionine permease6.9

Induction of genes involved in arginine biosynthesis

The synthesis of arginine in fungi has three main components, the synthesis of ornithine, the synthesis of carbamoyl phosphate and the conversion of the two compounds to arginine (Davis, 1986; Jauniaux et al., 1978). As shown in Table 2, eight of the 10 genes involved in arginine biosynthesis were upregulated more than two-fold after spermidine addition. In addition, ORT1, the gene involved in ornithine export from mitochondria was also induced.

Table 2. Genes induced by spermidine in the arginine, citrulline and ornithine metabolism*
Gene namesAnnoted functionsFold increase
  • *

    Data are average of five independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

ARG1Arginosuccinate synthetase, catalyses formation of L-argininosuccinate from citrulline and L-aspartate4.7
ARG2Acetylglutamate synthase, first step in the biosynthesis of the arginine precursor ornithine2
ARG3Ornithine carbamoyltransferase, catalyses the sixth step in ornithine biosynthesis3
ARG4Argininisuccinate lyase, this is the final step in arginine biosynthesis4.6
ARG5.6Processed in the mitochondrion to form acetylglutamate kinase and N-acetyl-γ-glutamyl-phosphate reductase, catalyses second and third steps in arginine biosynthesis2.8
CPA2Large subunit of carbamoyl phosphate synthetase, catalyses the synthesis of citrulline, an arginine precursor2.8
ECM40Mitochondrial ornithine acetyltransferase, catalyses the fifth step in arginine biosynthesis2.5
ORT1Ornithine transporter to the mitochondria, exports ornithine from mitochondria for arginine biosynthesis2

Induction of genes involved in biotin, lysine and tryptophan metabolism

Spermidine addition caused the upregulation of several genes involved in biotin, lysine and tryptophan metabolism (Table 3). All the yeast genes encoding biotin-synthesizing enzymes (BIO2, BIO3, BIO4), transporter of either biotin (VHT1) or precursor of biotin (BIO5), were upregulated by 10−5M spermidine addition (Table 3). A study of the time course of the induction of the BIO5 gene by real-time PCR showed that induction of this gene occurred as early as 2 h after the addition of the higher concentration of spermidine (Figure 3C).

Table 3. Genes induced by spermidine in the biotin, lysine and NAD (tryptophan) metabolism*
Gene namesAnnoted functionsFold induced
  • *

    Data are average of five independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

BIO5Putative transmembrane protein, responsible for uptake of 7-keto 8-aminopelargonic acid14.2
BIO3Adenosylmethionine-8-amino-7-oxononanoate terminase, biotin biosynthesis10.2
BIO4Dithiobiotin synthase activity, biotin biosynthesis9.9
BIO2Biotin synthase activity, biotin biosynthesis4.9
LYS9Saccharopine dehydrogenase (L-glutamate forming), lysine biosynthesis, aminoadipic pathway12
LYS1Saccharopine dehydrogenase (L-lysine forming), lysine biosynthesis, aminoadipic pathway10.1
LYS4Homoaconitase, catalyses the conversion of homocitrate homoisocitrate4.4
LYS2L-Aminoadipate-semialdehyde dehydrogenase, lysine biosynthesis, aminoadipic pathway3.7
LYS20Homocitrate synthase, lysine biosynthesis, aminoadipic pathway4.3
LYS12Homo-isocitrate dehydrogenase, lysine biosynthesis3
LYS21Homocitrate synthase isozyme, catalyses condensation of acetyl CoA and α-ketoglutarate to form homocitrate3.4
BNA2Tryptophan 2,3-dioxygenase, biosynthesis of nicotinic acid from tryptophan via kynurenine pathway11.1
BNA13-Hydroxyanthranilic acid dioxygenase, biosynthesis of nicotinic acid from tryptophan via kynurenine pathway, NAD biosynthesis3.4
BNA3Arylformamidase, biosynthesis of nicotinic acid from tryptophan via kynurenine pathway, NAD biosynthesis3.7
BNA4Kynurenine 3-mono oxygenase, biosynthesis of nicotinic acid from tryptophan via kynurenine pathway3
BNA5Kynureninase, required for de novo biosynthesis of NAD from tryptophan2.7

Eight genes involved in lysine biosynthesis from aminoadipic acid and homoisocitrate showed three-fold to 12-fold increased expression after the addition of spermidine. Several genes involved in tryptophan degradation were also induced by the addition of spermidine.

Induction of transport related genes after spermidine addition

Spermidine treatment for 6 h enhanced the expression of genes for several transporters, such as SUL1, SUL2 (involved in sulphate transport), BIO5 (a transporter of 7,8 aminopelargonic acid, a substrate of biotin biosynthesis), SEO1 (a putative permease member of the allantoate transporter family), CTR3 (which encodes the high-affinity copper transporter of the plasma membrane) and PHO84 (a gene encoding a high-affinity inorganic phosphate transporter) (Table 4). Real-time PCR analysis of several of these genes (BIO5, SEO1 and PHO84) showed that increased expression occurred 2 h after spermidine treatment (Figure 3). The genes for oligopeptide transporter OPT2 increased three-fold and gene expression of small peptide transporter PTR2 increased two-fold after spermidine treatment.

Table 4. Spermidine-induced genes involved in various transport functions*
Gene namesAnnoted functionsFold induced
  • *

    Data are average of five independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

SUL1Sulphate transporter activity, control the endogenous concentration of activated sulphur intermediates; PM7.8
BIO5Transmembrane regulator of KAPA/DAPA transport, permease activity, vitamin/cofactor transport; PM14.2
CTR3High-affinity copper transporter; PM2.7
SEO1Permease, suppressor of pulphoxyde ethionine resistance; PM5.2
GIT1Member of yeast sugar permease, phospholipids transporter activity; PM3.3
PHO84Inorganic phosphate transporter, phosphate metabolism; PM10.3
MMP1S-Methylmethionine permease, S-methylmethionine transport; PM6.9
SUL2Sulphate transporter activity, control the endogenous concentration of activated sulphur intermediates; PM4.6
OPT2Oligopeptide transporter; PM3
VHT1Vitamin H (biotin) transporter; PM4.1
MCH5Monocarboxylate permease, transporter activity; membrane4
FET4Low-affinity iron transporter; PM3.2
PHO89Na/Pi symporter, phosphate transport; PM2.4
PTR2Small peptide transport into the cell, peptide transporter2.1
MEP3Ammonia permease, ammonia transporter activity; PM2.2
TPO5Protein involved in polyamine transport2.1

Effect on transcription factors

Increased transcription of >240 genes may not reflect a direct effect of spermidine addition; spermidine may regulate altered expression of some of the transcription factors, which in turn control the expression of various genes induced by spermidine. Indeed, spermidine treatment resulted in increased expression of 16 transcription factors, as shown in Table 5. These include factors involved in amino acid metabolism (Met28p, Met32p, Gat1p) and inositol metabolism, as well as Zap1P, Hal9p, Tea1p that are involved in zinc regulation, increased salt tolerance and Ty1 enhancer activation, respectively.

Table 5. Spermidine-induced genes involved in encoding transcription factors*
Gene namesAnnoted function of encoded proteinFold change
  • *

    Data are avarage of five independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

MET28Transcriptional activator in the Cbf1p–Met4p–Met28p complex, regulator of sulphur metabolism5
MET32Zinc-finger DNA-binding protein, transcriptional regulation of methionine biosynthetic genes5
HIR1Transcriptional co-repressor, transcription of histone H2A, H2B, H3 and H4 genes2
ZAP1Zinc-regulated transcription factor, binds to zinc-responsive element, zinc ion homeostasis2.4
PHO4Basic helix–loop–helix transcription factor, regulated by phosphorylation, cellular response to phosphate starvation2.2
GAT1Regulation of nitrogen utilization, contains a GATA-1 type DNA-binding motif2.1
NRG2Negative regulator of glucose-controlled genes2.3
INO2Component of Ino2p/Ino4p basic helix–loop–helix transcription activator, required for derepression of phospholipid biosynthetic genes1.9
HAL9Zinc-finger transcription factor, overexpression increases salt tolerance1.5
WTM2WD repeat containing transcriptional modulator, involved in regulation of meiosis and silencing1.9
TEA1Ty1 enhancer activator, required for full levels of Ty enhancer-mediated transcription, zinc cluster DNA-binding protein1.5
TFC4One of six subunits of RNA polymerase III transcription initiation factor complex, part of TauA domain1.6

Gene expressions that are repressed after spermidine treatment

The repressed genes can be grouped into a few major categories; DNA, RNA binding, transcription (Table 6A) and stress-related genes (Table 6B). Several genes involved in encoding transcription factors, such as CIN5, MIG2, SUT1, GAT2 and STP4, are downregulated > two-fold. A few genes (such as HTL1, GIS1, ARP8 and SNF11) that were involved in the chromatin remodelling complex were downregulated. Also repressed were genes encoding pre-mRNA splicing (SNT309, ECM2), telomerase functions (HLT1) and genes involved in frameshift suppression (MBF1). Another major category of spermidine-repressed genes included various stress-responsive genes, such as HSP12, HSP104, GPD1, CYC7 and GPX2.

Table 6. Spermidine-repressed genes
Gene namesAnnoted functionsFold change
  • *

    Data are average of five independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

A. Spermidine-repressed genes involved in DNA, RNA binding and regulation of gene expression and splicing*
MBF1Transcriptional coactivator that bridges the DNA-binding region of Gcn4p and TATA-binding protein Spt15p, frameshift suppressor–3.1
GIS1JmjC domain-containing histone demethylase, transcription factor involved in the expression of genes during nutrient limitation−2.1
HTL1Component of the RSC chromatin remodelling complex, may function in chromosome stability, telomerase maintenance−1.9
RAD51Strand exchange protein, forms a helical filament with DNA that search for homology, involved in double-strand DNA break repair−1.8
MIG2Protein with zinc fingers, involved in repression of SUC2, multicopy inhibition of GAL gene expression−1.8
RPA135RNA polymerase I subunit A135, DNA-directed RNA polymerase activity−1.6
ARP8Nuclear actin-related protein involved in chromatin-remodelling enzyme complexes−1.9
SNF11Subunit of the SWI/SNF chromatin remodelling complex involved in transcriptional regulation−1.7
RSA1Protein involved in the assembly of 60S ribosomal subunits−1.7
GAT2Protein containing GATA family zinc-finger motifs, expression repressed by leucine−1.9
SNT309Component of NineTeen complex, involved in RNA splicing−2
STP4Protein containg a Kruppel-type zinc-finger domain−2.4
MED7Subunit of the RNA polymerase II mediator complex, essential for transcriptional regulation−2.1
CIN5Basic leucine zipper transcriptional factor of yAP-1 family, involed in drug resistance and salt tolerance−2.4
TOF2Topoisomerase I-interacting factor, may be involved in DNA topological changes−1.8
SUT1Transcription factor of the zinc family, involved in induction of hypoxic gene expression−2
ECM2Pre-mRNA splicing factor−1.8
NPL3RNA-binding protein, carries poly(A)+ mRNA from nucleus into the cytoplasm, phosphorylated by Sky1p−1.8
B. Spermidine-repressed genes involved in various stress responses*
HSP12Plasma membrane-localized protein, induced by heat shock, oxidative stress, osmotic stress, glucose depletion−2.8
HSP104Responsive to stress (heat, ethanol and sodium arsenite), involved in [PSI+] propagation−3.4
HAP4Subunit of the haem-activated, glucose-repressed Hap-protein complex, a transcriptional activator of respiratory gene expression−3
GPD1NAD-dependent glycerol-3-phosphate dehydrogenase, essential for growth under osmotic stress−2.8
ROX1Haem-dependent repressor of hypoxic genes, responsible for DNA bending activity−3.4
GAD1Glutamate decarboxylase, converts glutamate into γ-aminobutyric acid, involved in response to oxidative stress−3.4
SDP1Stress-inducible dual-specific MAP kinase phosphatase−2.7
CYC7Cytochrome c isoform 2, expressed under hypoxic condition−5.3
CTT1Cytosolic catalase T, has a role in protection from oxidative damage by hydrogen peroxide−6.6
GPX2Glutathione peroxidase, induced by glucose starvation, protects cells from phospholipids hydroperoxides and non-phospholipid peroxides during oxidative stress−1.8

Effect of spermine addition on global gene expression

An important part of this study concerns the differential effects of spermidine and spermine on gene expression. In all of the above experiments, part of the administered spermidine was converted by the cell to spermine by spermine synthase (SPE4; data not shown). To differentiate the effects due to spermidine from those due to spermine, we also carried out a parallel study on the effects of added spermine on gene expression. In the double mutant that we used (spe3Δ fms1Δ), the added spermine could not be converted to spermidine as this strain lacked spermine oxidase (as in the other experiments, all of these cultures also contained 10−8M spermidine, since, as we have already shown, spermine does not permit growth of this spermidine auxotroph; Chattopadhyay et al., 2003).

As shown in Table 7, as opposed to the results found after spermidine addition, spermine addition at 10−5M concentration resulted in a change in the expression of very few genes. Some of the spermine-induced genes were also induced by spermidine, as indicated in Table 7; however, their level of induction was far below than that found after spermidine addition. Only 15 genes were induced two-fold, and three genes were repressed two-fold by spermine.

Table 7. Spermine-controlled genes*
GenesymbolFold-changeAnnoted function
  • *

    Data are average of three independent experiments (p < 0.05); functional annotations are based on Saccharomyces Genome Database (www.yeastgenome.org).

  • Also changed by spermidine addition.

Up-regulated genes
CPA22.7Large subunit of carbamoyl phosphate synthetase, synthesis of citrulline, the arginine precursor
ARG42.3Argininosuccinate lyase, arginine biosynthesis
YKL218C (SRY1)2.23-Hydroxyaspartate dehydrogenase, serine racemase
RPL7B2.5Protein component of the 60S ribosomal subunit
ECM402Mitochondrial ornithine acetyltransferase, ornithine biosynthesis
HIS51.7Histidinol-phosphate aminotransferase, histidine biosynthesis
BNA12.23-Hydroxyanthranilic acid dioxygenase, biosynthesis of nicotinic acid from tryptophan
MEP32Ammonium permease, expressed under nitrogen catabolite repression
MET131.6Isozyme of methylenetetrahydrofolate reductase, methionine biosynthesis
LYS11.9Saccharopine dehydrogenase, lysine biosynthesis
SSU11.7Plasmamembrane sulphite pump, efficient sulphite efflux
MCH41.6Monocarboxylate transporter homologue, transport of monocarboxylic acid across the plasma membrane
YEL073C2Uncharacterized protein, regulated by inositol/choline
Down-regulated genes
HHO1−1.7Histone H1, suppresses DNA repair involving homologous recombination
PCL1−1.6Ph085 cyclin, involved in entry into the mitotic cell cycle
CLN2−1.7G1 cyclin involved in regulation of cell cycle, promotes G1–S phase transition
SNF11−1.4Subunit of the SWI/SNF chromatin remodelling complex involved in transcriptional regulation
BBP1−1.4Spindle pole body duplication, required for mitotic function of Cdc5p
SRL1−1.5Mannoprotein exhibiting tight association with cell wall, suppressor of Rad53p null lethality
CWC25−1.5Component of a pre-mRNA splicing complex containing Cef1p

Discussion

In this paper we have reported the first global microarray analyses of the effect of added spermidine and spermine in a eukaryotic system. In particular, we have studied these effects in a system designed to minimize any effects of these additions on the growth rate of the cultures; namely, by comparing cultures grown in 10−5M spermidine and spermine with cultures grown in 10−8M spermidine. As seen in Figure 1, the growth rate was only slightly faster in the cultures grown with 10−5M spermidine and was not affected at all by the addition of 10−5M spermine.

We have recently reported that yeast cells grown at a nearly optimum growth rate in the presence of 10−8M spermidine; >50% of the spermidine was used for hypusine modification of eIF5A; so it was clear that one of the major functions of spermidine is the modification of eIF5A (Chattopadhyay et al., 2008). A major stimulus for the current studies was the question of why wild-type S. cerevisiae cells normally contain a much higher internal concentration of spermidine than needed for optimum growth (1000-fold). Hence, it was interesting to note that in the current study so many genes were up- or downregulated after spermidine addition, even though there was little effect on the growth rate. A number of different systems were affected, as shown by the data in Tables 1–6 and Figure 2, and some of the changes were very large. Particularly striking were the effects on most of the genes involved in sulphur metabolism and on methionine transport and biosynthesis, as well as arginine and lysine and biotin biosynthesis. In these microarray studies, we have found increased expression of Met32p and Met28p by spermidine, which constitute the main transcription activators of the sulphate assimilation pathway, including Cbf1p, Met4p and Met31p (Kuras et al., 1996; Blaiseau et al., 1997).

Another interesting change was in the cluster of genes involved in biotin biosynthesis, such as BIO5, BIO3 and BIO4 (Phalip et al., 1999) along with BIO2; all these genes were induced by spermidine addition. The genes for biotin biosynthesis in yeast are present as a gene cluster on chromosome XIV and are regulated by environmental stress (Gasch et al., 2000), iron deprivation (Shakoury-Elizeh et al., 2004), glucose limitation (Ferea et al., 1999) or histone modification (Wyrick et al., 1999). Biotin is essential for all living organisms and is a cofactor for several of the carboxylase family of enzymes. Saccharomyces cerevisiae is auxotrophic to biotin; however, it can be complemented by the addition of 7-keto 8-aminopelargonic acid (KAPA), 7,8-diaminopelargonic acid (DAPA) or dithiobiotin to the medium. Biotin biosynthesis is also increased by S-adenosylmethionine, which serves as an amino group donor in the synthesis of KAPA from DAPA (Fontecave et al., 2004). Spermidine addition upregulated genes in methionine biosynthesis, including the synthesis of S-adenosylmethionine; on the other hand, spermidine treatment also repressed S-adenosylmethionine decarboxylase (SPE2) by 1.5-fold (see below). Thus, it is possible that the accumulated S-adenosylmethionine can trigger the increased expression of biotin-biosynthesizing genes as an indirect effect of spermidine treatment.

Surprisingly, some of the genes induced by spermidine treatment (especially those involved in methionine metabolism) were found by Aranda and del Olmo (2004) to be induced by acetaldehyde treatment. They also found that acetaldehyde treatment resulted in the induction of polyamine transport genes (TPO2, TPO3). Other genes involved in vitamin B1 biosynthesis and in aryl alcohol metabolism were also induced by both acetaldehyde treatment and spermidine addition (Aranda and del Olmo, 2004) (Table 3; and for complete microarray data, see GEO Accession No. GSE15269). In a different study, Santiago and Mamoun (2003) observed that several genes involved in inositol, methionine and biotin biosynthesis were downregulated, along with genes implicated in polyamine transport (TPO1, TPO2) by addition of inositol or choline to yeast cultures. Although we have no explanation for this similarity in the effects of these three very different treatments, it seems possible, from these global gene-expression studies and our current study, that polyamine levels might play a role in the regulation of these gene expressions.

Our microarray data showed 1.5-fold downregulation of S-adenosylmethionine decarboxylase (SPE2) gene expression after spermidine treatment. Expressions of the known polyamine transporters were unchanged except for TPO5, which showed a 2.1-fold induction of gene expression after spermidine addition. There was no change in gene expression of ornithine decarboxylase (SPE1) after spermidine or spermine treatment, which suggests a post-translational control of ornithine decarboxylase by antizyme (Palanimurugan et al., 2004).

The large number of very different systems affected by the addition of spermidine emphasizes the importance of the higher internal concentration of spermidine normally present in wild-type yeast cells. However, at this time we are unable to define which of the systems represent the primary effect of the addition of spermidine or the results of indirect effects of other gene expression. Real-time PCR analyses of five of the induced genes (Figure 3B, C) suggest that spermidine addition may result in an indirect effect on gene expression of various pathways, as most of the gene expressions were changed after 2 h of spermidine addition. Of particular interest is the increased expression of a number of transcription factors after spermidine addition, particularly those involved in the expression of several genes in methionine, arginine and other amino acid-metabolizing genes (Table 5). A study by Yoshida et al. (2004) in E. coli has postulated the involvement of polyamine modulation in the expression of genes responsible for bacterial growth (Yoshida et al., 2004; Igarashi and Kashiwagi, 2006). In their experiments they showed that most of the genes enhanced by polyamines were not under the direct control of polyamines, but were due to the indirect effect of transcription factors whose synthesis was enhanced by polyamines. Thus, it seems possible that increased transcription of various metabolic pathways might be regulated indirectly by change in the transcription factors, whose expressions are high due to the higher concentration of spermidine.

Some of the genes repressed by spermidine also include genes in nucleic acid function (Table 6A), which may be due to binding of an excess amount of spermidine to the negatively charged nucleic acids (Cohen, 1998, and references therein; Igarashi and Kashiwagi, 2000; Vijayanathan et al., 2001). Spermidine and spermine repressed the SWI–SNF chromatin-remodelling complex of yeast (Tables 6A, 7). Polyamine involvement in the regulation of gene expression through the modulation of the chromatin remodelling complex has been demonstrated in yeast and other systems (Pollard et al., 1999; Huang et al., 2007).

Yeast polyamine mutants grown in 10−8M spermidine did not show any obvious defect in growth or any indication of oxidative stress (Chattopadhyay et al., 2006) when the spermidine level was restored to wild-type level by adding 10−5M spermidine to the culture medium; various stress-responsive genes were repressed; such as HSP12, HSP104, ROX1, CYC7 and others (Table 6B).

Our study provides new insights into the responsiveness of yeast mutants lacking spermidine synthase to the millimolar level of polyamines, which are present in wild-type yeast cells and may suggests specific molecular targets of the high intracellular concentration of spermidine that is normally present in wild-type yeast. More analyses and experimental data, as well as comparable studies with different yeast mutants, are needed to distinguish between the direct and indirect effects of the polyamines and to explain the physiological connections between the different pathways affected by spermidine. Moreover, these results are complicated by the fact that when spermidine concentration is in excess (10−5M), the modified eIF5A level is >20-fold that needed for optimum growth (Chattopadhyay et al., 2008). In addition, it is likely that the added spermidine would repress the enzymes involved in polyamine biosynthesis, with a resultant decrease in the level of intracellular putrescine and decarboxylated S-adenosylmethionine, and that these changes might affect other systems involved in arginine and methionine metabolism. In conclusion, even though it is not possible to define with certainty all of the direct effects of spermidine, the data in this paper clearly indicate that spermidine has a profound effect on the expression of a large number of genes, either directly or indirectly.

Acknowledgements

This research was supported by the Intramural Research Programme of the NIH (National Institute of Diabetes, Digestive and Kidney Diseases).

Ancillary