In this paper we have reported the first global microarray analyses of the effect of 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 10−5 M of spermidine and spermine with cultures grown in 10−8 M spermidine. As seen in , the growth rate was only slightly faster in the cultures grown with 10−5 M spermidine, and was not affected at all by the addition of 10−5 M spermine.
Recently, we have reported that yeast cells grown at a nearly optimum growth rate in the presence of 10−8
M spermidine and >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 regulated or down regulated 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 – and , and some of the changes were very large. Particularly striking were the effects on most of the genes involved in sulfur 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 sulfate 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
(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 carboxylase family of enzymes. Saccharomyces cerevisiae
is auxotrophic to biotin, however, can be complemented by addition of KAPA 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 the 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 Olmo (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
). Other genes involved in vitamin-B1 biosynthesis and in aryl alcohol metabolism were also induced both by acetaldehyde treatment, and by spermidine addition (Aranda and del Olmo, 2004
), ( and for complete microarray data see GEO accession NO GSE15269). In a different study, Santiago and Mamoun (Santiago and Mamoun, 2003
) observed that several genes involved in inositol, methionine, and biotin biosynthesis were down-regulated along with genes implicated in polyamine transport (TPO1
) 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 expression.
Our microarray data showed 1.5-fold down regulation of S-adenosylmethionine decarboxylase (SPE2
) gene expression after spermidine treatment. Expressions of the known polyamine transporters were unchanged except 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 () suggest the notion that spermidine addition may result in an indirect effect on gene expression of various pathways, as most of the gene expressions were changed after 2h 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 (). A study by Yoshida et al (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 experiment they have shown that most of the genes enhanced by polyamines were not under the direct control of polyamines, but were due to 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 (), which may be due to binding of excess amount of spermidine to the negatively charged nucleic acids (Cohen SS, 1998
, and references there in; Igarashi and Kashiwagi, 2000
; Vijayanathan et al., 2001
). Spermidine and spermine repressed the SWI-SNF chromatin-remodeling complex of yeast (, ). Polyamine involvement in the regulation of gene expression through the modulation of chromatin remodeling complex has been demonstrated in yeast and other systems (Pollard et al., 1999
; Huang et al., 2007
Yeast polyamine mutants grown in 10−8
M 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 10−5
M spermidine to the culture medium, various stress responsive genes were repressed such as HSP12
, and others ().
Our work provides new insights into the responsiveness of yeast mutant lacking spermidine synthase to 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 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 (10−5
M), the modified eIF5A level is more than 20-fold than 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.