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Department of Microbiology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The IMP dehydrogenase 2 (IMD2) gene provides a good model system to examine transcriptional regulation because its expression is regulated at several steps during transcription. The IMD2 gene encodes a rate-limiting enzyme for de novo synthesis of GTP. Several cis-elements involved in the regulation of IMD2 gene expression have been identified. One is located in the protein-coding region of the IMD2 gene, and functions to repress IMD2 gene expression when nutrition is limited (Escobar-Henriques et al. 2003a). IMD2 gene expression is also modulated by the cell GTP concentration. Expression of the IMD2 gene is decreased or increased by the addition of guanine or by treatment with the GTP-depleting drugs mycophenolate (MPA) or 6-azauracil (6-AU), respectively (Shaw & Reines 2000; Escobar-Henriques et al. 2003b; Hyle et al. 2003). GTP concentration-responsive regulation requires the guanine response element (GRE) located in the IMD2 gene promoter (Escobar-Henriques & Daignan-Fornier 2001; Escobar-Henriques et al. 2003b). Recently, an interesting model was proposed for GTP-dependent IMD2 gene expression (Davis & Ares 2006; Steinmetz et al. 2006). Under higher GTP concentrations, novel transcription start sites, located approximately 100 base pairs upstream of the transcription start site used under lower GTP concentration, were discovered. Transcription from these upstream transcription start sites is immediately terminated in a Sen1 termination factor-dependent manner, resulting in the production of short transcripts (approximately 250 base pairs). A rough estimation of the short transcripts size and the presence of RNA polymerase II suggest that transcription termination occurs around the translation initiator ATG codon. This termination may involve the repressive element (RE) which is located downstream of the novel transcription start sites (Shaw et al. 2001; Kopcewicz et al. 2007). The RE is a potent repressor because when placed downstream of the GAL1 promoter, Gal4-dependent transcription is abolished. Furthermore, mutation in this region causes increased IMD2 gene expression (Kopcewicz et al. 2007). This termination is also mediated by another transcription terminator-related protein, Rna15 (Kopcewicz et al. 2007). This termination is bypassed under GTP-depletion conditions, because of a shift to the downstream transcription start site. As a result, full-length mRNAs are produced. The factors involved in the shift of this transcription start site are not yet known.
Several transcription elongation factors, such as S-II/TFIIS and Rpb9, are required for IMD2 gene induction by MPA or 6-AU treatment (Shaw & Reines 2000; Desmoucelles et al. 2002; Ubukata et al. 2002). S-II and Rpb9 relieve RNA polymerase II from transcription elongation arrest, which is caused by obstacles in the template DNA, such as DNA binding proteins (Reines et al. 1989; Reines & Mote 1993; Awrey et al. 1997). The proteins that repress IMD2 gene expression must be determined to further understand the induction mechanisms mediated by transcription elongation factors.
A genome-wide screen of MPA sensitivity performed by Desmoucelles et al. (2002) demonstrated that mutations in components of the transcriptional co-activator SAGA complex frequently induce MPA and 6-AU sensitivity. One of these components is Spt8, which interacts physically and functionally with S-II (Wery et al. 2004): mutations in both the DST1 and SPT8 genes result in MPA hypersensitivity. Mutations in Mediator, another transcriptional co-activator complex, also increase MPA or 6-AU sensitivity: the srb5–77 mutation causes 6-AU sensitivity when combined with the RTF1 gene null mutation (Costa & Arndt 2000) and disruption of GAL11 gene causes both MPA and 6-AU sensitivity (Riles et al. 2004). Moreover, the dst1
srb5 mutant has a growth defect under normal conditions (30 °C) (Malagon et al. 2004). These findings suggest that these transcriptional co-activators have important roles in IMD2 gene expression. Indeed, yeast cells bearing mutations in components of the SAGA complex, such as Spt8, Spt3 and Spt20, are defective in IMD2 gene expression, probably as a result of its role in transcription elongation (Desmoucelles et al. 2002). In contrast, although Sub1 is a traditional transcriptional co-activator, it is not known whether Sub1 itself affects MPA resistance and IMD2 gene expression, or whether Sub1 is functionally linked with other transcription factors involved in MPA resistance, like S-II.
Sub1 functions during multiple transcription steps in Saccharomyces cerevisiae, including initiation, elongation and termination. Sub1 was originally identified as a transcriptional stimulator in vitro (Henry et al. 1996; Knaus et al. 1996). It was also identified genetically as a suppressor of a mutation in the transcription initiation factor TFIIB (Knaus et al. 1996). Over-expression of the SUB1 gene suppresses cold-sensitive growth in the TFIIB mutant. In addition, TFIIB mutations are synthetically lethal with a null mutation of the SUB1 gene. Furthermore, over-expression of the SUB1 gene promotes transcription dependent on the Gcn4 or Hap4 activator in vivo, indicating that Sub1 functions as a transcriptional co-activator at the transcription initiation step (Knaus et al. 1996). Recent studies by Calvo and Manley provided evidence for novel roles of Sub1: Sub1 interacts with the transcription machinery throughout the transcription elongation and termination steps as well as the initiation step, suggesting a role of Sub1 in multiple steps of transcription (Calvo & Manley 2005). Consistent with this, Sub1 functions in transcription termination processes through its interaction with the transcription termination factor Rna15, which results in a change in the termination position (Calvo & Manley 2001). PC4, a homologue of Sub1 in mammalian cells, has a role as a co-activator (Ge & Roeder 1994; Kretzschmar et al. 1994). PC4 is involved in transcription termination of RNAPIII and its involvement in RNAPII transcription termination has also been discussed (Wang & Roeder 1998; Calvo & Manley 2001). The highly conserved regions between Sub1 and PC4 include the single strand DNA (ssDNA) binding domain and the serine-rich domain (Calvo & Manley 2001). The roles of these domains of Sub1 in transcription regulation, however, are not yet clear.
In the present study, disruption of the SUB1 gene increased IMD2 gene expression under normal growth conditions, even in the absence of S-II, suggesting that Sub1 functions to retain the repressed state of IMD2 gene expression. This finding implies a novel role for Sub1 during transcription: Sub1 functions to repress the expression of some genes.
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Results |
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To evaluate whether Sub1 is functionally linked with S-II, we constructed a dst1 sub1 double-disrupted yeast. The double-disrupted mutant yeast had no apparent defects in cell growth under general laboratory conditions (Fig. 1A). We then assessed the effect of SUB1 gene disruption on the drug sensitivities of the dst1 mutant.
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sub1 cells in the presence of MPA; dst1 sub1 was more resistant to MPA than dst1, but only slightly sensitive to MPA compared with the wild-type strain (Fig. 1A). Thus, SUB1 gene disruption partially suppressed the MPA sensitivity of dst1. Disruption of the SUB1 gene in the presence of the DST1 gene, however, led to no detectable change in MPA resistance. Additionally, the introduction of plasmids bearing the SUB1 gene to dst1 sub1 increased sensitivity to MPA. Furthermore, SUB1 gene over-expression in dst1 caused MPA hypersensitivity. These findings indicate that Sub1 genetically interacts with S-II.
We also evaluated the contributions of Sub1 to dst1 sensitivity to drugs other than MPA. dst1 is sensitive to the oxidant menadione, caffeine and the microtubule-destabilizing drug thiabendazole, and so on (Koyama et al. 2003; Malagon et al. 2004; Wery et al. 2004). SUB1 gene disruption or over-expression had a slight or no detectable effect on dst1 sensitivity to these drugs (data not shown).
We then examined whether Sub1 genetically interacts with another transcription elongation factor, Rpb9 (Hemming et al. 2000). Rpb9, a subunit of RNA polymerase II, confers yeast resistance to MPA (Van Mullem et al. 2002). rpb9 cells show a growth defect under normal culture conditions. We confirmed that the rpb9 cells used in this study exhibited a slight growth defect (data not shown). Disruption of the SUB1 gene suppressed the growth defect of rpb9 cells when treated with MPA (Fig. 1B), suggesting that disruption of the SUB1 gene rescues the defect in transcription elongation caused by RPB9 gene disruption.
Sub1 represses IMD2 gene expression
To determine the role of Sub1 in drug sensitivity, we analyzed IMD2 gene expression, which is critical for MPA resistance (Shaw et al. 2001). IMD2 gene expression is induced in response to a reduction in the GTP pool provoked by MPA treatment, and this induction requires both S-II and Rpb9 (Shaw & Reines 2000; Desmoucelles et al. 2002). That is, IMD2 gene expression is not induced by MPA treatment in DST1 or RPB9 gene disruptants (Fig. 2A; Shaw & Reines 2000; Desmoucelles et al. 2002). Consistent with a previous report (Shaw et al. 2001), dst1
sensitivity to MPA was suppressed by over-expression of the IMD2 gene (Fig. 2B). We hypothesized that SUB1 gene disruption increases IMD2 gene expression. To investigate this issue, we examined IMD2 gene expression in dst1 sub1 cells using Northern blot analysis. IMD2 gene expression in dst1 sub1 was higher than that in dst1 in the presence of MPA (Fig. 2A). Of note, the IMD2 gene was expressed in dst1 sub1 even in the absence of MPA. sub1 also exhibited increased IMD2 gene expression in the absence of MPA compared with wild-type cells. These results strongly suggest that Sub1 functions to repress IMD2 gene expression. In contrast, the IMD2 gene expression level in sub1 in the presence of MPA was lower than that in the wild-type strain (Fig. 2A, left image). It is possible that the effect of the previously described elongation stimulatory function of Sub1 (Calvo & Manley 2005) contributes to IMD2 gene expression especially under MPA (+) conditions.
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sub1 cells was less than that in wild-type or in dst1
cells (Fig. 2C). Thus, the GTP concentration was maintained in dst1 sub1 cells, even in the presence of MPA, probably because of increased IMD2 gene expression. PC4-like region of Sub1 is sufficient to repress IMD2 gene expression
We next determined the region of Sub1 required to repress IMD2 gene expression. Sub1 contains three domains: a serine-rich domain, an ssDNA binding domain and a carboxyl-terminal region (Calvo & Manley 2001). The serine-rich and the ssDNA binding domains are conserved in the mammalian homologue PC4. To identify the region required for IMD2 gene repression, we constructed partially deleted mutant SUB1 genes (Fig. 3A). When plasmids encoding the wild-type SUB1 gene were introduced, the IMD2 gene expression level was reduced in dst1 sub1 (Fig. 3B). Introduction of a mutant lacking the carboxyl-terminal region del-1 or del-3 also reduced IMD2 gene expression levels, suggesting that the carboxyl-terminal region was not essential. Importantly, however, the PC4-like region containing the serine-rich domain and the ssDNA binding domain was sufficient for Sub1 to exert its repression function. In addition, IMD2 gene expression was reduced in the del-2 mutant in which the serine-rich domain was deleted, compared with cells bearing the empty vector. Therefore, the serine-rich domain was dispensable for the repressive function. We also tested a Sub1 mutant that lacked the ssDNA binding domain as well as the serine-rich domain. Unfortunately, expression of this mutant protein fused to the FLAG tag was quite reduced compared with that of the wild-type protein, based on Western blot analysis for the FLAG tag (data not shown). Thus, we could not determine the contribution of the ssDNA binding domain to IMD2 gene expression, although this mutant failed to decrease IMD2 gene expression.
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sub1 cells (Fig. 3C), but altered the IMD2 gene expression level (Fig. 3B). Thus, Sub1 has a function to control MPA sensitivity, which is different from the function required for IMD2 gene regulation. Moreover, this function is carried in the Sub1's carboxyl-terminal region which is not conserved in PC4, suggesting that this function is specific for yeast Sub1. In addition, del-2 led to MPA sensitivity, indicating that the serine-rich region is dispensable for the regulation of MPA sensitivity. Sub1 localization around the IMD2 gene promoter
We next determined the location of the Sub1 proteins in the IMD2 gene. We carried out chromatin immunoprecipitation (ChIP) analyses using wild-type cells expressing FLAG-tagged Sub1 proteins (Fig. 4). Consistent with a previous report (Calvo & Manley 2005), high amounts of Sub1 proteins were detected in the ACT1 promoter, whereas only small amounts of Sub1 proteins were detected in the non-transcribed gene HMR1. In the non-induction condition MPA (–), large amounts of Sub1 proteins were detected around the IMD2 promoter region, whereas there was little Sub1 around the open reading frame (ORF) (Fig. 4). These results suggest that Sub1 functions around the promoter region of the IMD2 gene under the non-induction condition. In the induction condition MPA (+), Sub1 proteins were present on the IMD2 ORF as well as in the promoter region, consistent with a previous report describing the interaction of Sub1 with RNA polymerase II through the transcription process including initiation, elongation and termination (Calvo & Manley 2005).
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Gene reporter techniques were used to determine whether the region upstream of the protein coding region in the IMD2 gene including the promoter is involved in the Sub1-mediated repression. Previous studies demonstrated that the presence of some regulatory cis-elements in the region upstream of the IMD2 protein coding region, as well as in the protein coding region itself (Escobar-Henriques & Daignan-Fornier 2001; Shaw et al. 2001; Escobar-Henriques et al. 2003a,b). We constructed a reporter plasmid bearing the region upstream of the IMD2 protein coding region, followed by the lacZ reporter gene (Fig. 5A, Reporter #1). Consistent with the data on IMD2 gene mRNA levels (Fig. 2A), β-galactosidase activity was higher in sub1 cells and dst1 sub1 cells harboring the Reporter #1 plasmid than in wild-type or dst1
cells harboring the reporter plasmid in the absence of MPA (Fig. 5B, black bar). This finding indicated that the increased IMD2 gene expression caused by SUB1 gene disruption was mediated by the region upstream of the protein coding region, including the promoter. Furthermore, the use of another reporter plasmid bearing approximately 70% of the IMD2 protein coding region as well as the region upstream of the IMD2 protein coding region revealed that dst1 sub1 cells contained higher β-galactosidase levels than wild-type or dst1 cells in the absence of MPA (data not shown). Thus, the contribution of the IMD2 protein coding region to Sub1-mediated repression may be insignificant, whereas Sub1 exerts its transcriptional repressive function in the region upstream of the IMD2 protein coding region.
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cells harboring the reporter plasmid, MPA treatment increased β-galactosidase activity, which did not coincide with the mRNA data shown in Fig. 2A. Escobar-Henriques et al. also described that no effect of S-II was observed in a reporter assay bearing only the region upstream of the protein coding region (Escobar-Henriques et al. 2003b). These results suggest that S-II functions in transcription of the protein coding region, which is consistent with the fact that the transcription elongation function of S-II is considered to be responsible for the IMD2 gene expression (Ubukata et al. 2002). In dst1 sub1 cells harboring the reporter genes, increased β-galactosidase activity was not detected following the addition of MPA. Short transcript production is decreased by SUB1 gene disruption
To further analyze the function of Sub1 around the region upstream of the IMD2 protein coding region, we focused on the previously proposed two transcription start sites model of IMD2 gene expression (Davis & Ares 2006; Steinmetz et al. 2006). In this model, transcription is initiated from the upstream transcription start sites under the non-induction condition, producing short transcripts because of premature termination probably around the translation initiator ATG codon, and resulting in the repression of IMD2 gene expression. In contrast, transcription is initiated from the downstream transcription start site under the induction condition, which bypasses the transcription termination. The short transcripts are not easily detected because of their rapid degradation by the nuclear exosome. Therefore, we used cells bearing Reporter #1 which is a multi-copy plasmid of the IMD2 gene. Short transcripts accumulated in the dst1 cells, whereas they were almost undetectable in dst1 sub1 cells (Fig. 5C). The downstream transcription start site shift occurred under low GTP concentrations (Davis & Ares 2006; Steinmetz et al. 2006). Indeed, the short transcript levels in the dst1 cells were decreased under the MPA (+) condition (Fig. 5C). These results suggest that Sub1 is involved in short transcript production, resulting in the repression of IMD2 gene expression.
Mutations in the GRE but not the RE diminished the effect of Sub1-mediated repression
To further examine the role of Sub1 around the IMD2 promoter region, we attempted to identify the precise region required for Sub1 repression of IMD2 gene expression. We constructed four reporter plasmids (Fig. 5A, Reporters #2–5): Reporter #2 lacked the RE, Reporter #3 had point mutations on the RE corresponding to the 1651 mutation (Escobar-Henriques et al. 2003b), Reporter #4 lacked the region between the GRE and RE, and Reporter #5 had point mutations on the GRE corresponding to the 1649 mutation (Escobar-Henriques et al. 2003b). Disruption of the SUB1 gene in wild-type and dst1 cells increased IMD2 gene expression in the Reporter #2 or 3-bearing cells, but not in the Reporter #4 or 5-bearing cells (Fig. 5D–G; black bars: MPA (–)). The ratios of the expression levels between wild type and sub1, or between dst1 and dst1 sub1, are shown in Fig. 5H. Under the MPA (–) condition (Fig. 5H, black bars), the Sub1 repression of reporter gene expression was still observed in the Reporter #2 or 3-bearing cells, although the effect was partially decreased in the Reporter #2-bearing cells (compare column 2 with column 1 in Fig. 5H, black bars). In contrast, the effect was completely blocked in the Reporter #4 or 5-bearing cells (the ratio was nearly 1.0). Thus, the GRE and the intermediate region between the GRE and the RE are indispensable for observing the effect of Sub1 on the IMD2 gene expression. These results suggest that Sub1 functions to repress IMD2 gene expression via the GRE and the intermediate region between the GRE and the RE.
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Discussion |
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Transcription repression mechanism mediated by Sub1
Our findings indicated that Sub1 is essential for the repression of IMD2 gene expression under normal growth conditions. The GRE and the intermediate region between the GRE and the RE were essential to reproduce the repressive effect of Sub1. The short transcripts assay and the ChIP analysis also provided evidence consistent with a role for Sub1 near the IMD2 promoter region.
How does Sub1 repress IMD2 gene expression? The two transcription start sites mechanism is important in IMD2 gene expression. With this model, the IMD2 gene is regulated by the combination of the transcription start site selection and the premature termination of transcription initiated from the upstream transcription start sites under non-induction conditions (Steinmetz et al. 2006). This termination may be induced by the RE (Kopcewicz et al. 2007). We found that deletion of the RE terminator partially decreased, but did not completely block the Sub1-dependent IMD2 gene expression (Fig. 5H, black bars 1 and 2). Therefore, even if Sub1 functioned in RE-dependent termination, this function would not be sufficient to account for the repressive effect of Sub1. Another cis-element to be considered is the GRE, because mutations in the GRE enhanced the use of the downstream transcription start site and full-length transcript production, even under the non-induction condition (Escobar-Henriques et al. 2003b). This latter phenomenon is analogous to the sub1 cell phenotype. One possible scenario is that disruption of the SUB1 gene changes the transcription start site from the upstream regions to the downstream regions. If so, the number of short transcripts that derive from the upstream transcription start sites would also be decreased. Furthermore, the effect of Sub1 would be diminished in cells with GRE mutations. Deletion of the intermediate region between the GRE and the RE may enhance the use of the downstream start site, because this deleted region contains the upstream start sites. Thus, this deletion can lead to a loss of the Sub1 effect. Furthermore, deletion of the RE would reduce the effect of Sub1; deletion of the RE would not essentially affect the expression level in sub1, but would increase it in wild type, because transcription from the upstream start sites, which is sensitive to the RE, would occur only in wild-type cells but not in sub1 cells. Thus, it is possible that disruption of the SUB1 gene causes a downstream shift of IMD2 gene transcription start site. In other words, Sub1 might retain the transcription start sites at the upstream region in the IMD2 gene.
Previously, Sub1 was suggested to be involved in transcription start site selection (Wu et al. 1999). Sub1 genetically interacts with some TFIIB mutants that have defects in transcription start site selection (Knaus et al. 1996; Wu et al. 1999). These TFIIB mutations lead to a downstream shift of the transcription start sites of the ADH1 and CYC1 genes, and also cause cold-sensitive growth (Pinto et al. 1992). Over-expression of Sub1 suppresses this cold-sensitive growth, whereas disruption of the SUB1 gene is synthetic-lethal (Knaus et al. 1996). Although there is no direct evidence that Sub1 alters the transcription start site (Wu et al. 1999), these findings raise the possibility that Sub1 retains the upstream transcription start site. If so, disruption of the SUB1 gene would lead to a downstream shift of the transcription start site, which would account for the present data.
During revision of the present paper, Rpb9 was proposed to be a transcription start site determinant of the IMD2 gene (Jenks et al. 2008). A mutation of the RPB9 gene suppressed the transcription start site defects in TFIIB mutants (Sun et al. 1996). In our study, the MPA sensitivity of rpb9 was suppressed by SUB1 gene disruption. Therefore, the transcription start site defects in rpb9 cells might also be suppressed by SUB1 gene disruption. Future experiments to analyze whether the IMD2 gene transcription start site selection is influenced by Sub1 or by its interplay with Rpb9 and TFIIB will provide valuable information.
Roles of S-II and Sub1 in IMD2 gene expression
The role of Sub1 in IMD2 gene expression is to maintain a repressed state under non-induction conditions. The results of the lacZ reporter gene assay indicated that the region upstream of the IMD2 protein coding region is responsible for Sub1-mediated repression. The reporter gene expression from this region is induced by MPA even in the absence of the DST1 gene, suggesting that S-II is not essential for relief of the transcriptional repression caused by Sub1 in vivo.
Unexpectedly, deletion of the DST1 gene increased IMD2 gene expression only in the absence of the SUB1 gene under the MPA (–) condition, although S-II is widely accepted as a transcription stimulator. It might be that the repressive effect of Sub1 is inhibited by S-II in the absence of MPA. These findings imply a functional relationship between Sub1 and S-II. In addition, there might also be another repressor, because the IMD2 gene expression level in sub1 cells under the MPA (–) or MPA (+) condition did not reach the fully induced state of wild-type cells under the MPA (+) condition. Further analyses are required to fully understand the regulation, although based on the suppression of the dst1 cell phenotypes the repressive function of Sub1 should be the central contributor.
Conserved function of Sub1 in transcription repression
PC4, a homologue of Sub1 in mammalian cells, has repressive functions that have been dissected by in vitro transcription systems. The present study revealed that only the PC4-like region of Sub1 (the serine-rich and the ssDNA binding regions) is sufficient for IMD2 gene regulation, raising the possibility that the repressive function of Sub1 is similar to that of PC4. PC4 seems to have several modes of repression, conferred by a topologic effect on transcribed DNA via its ssDNA binding activity (Werten et al. 1998; Fukuda et al. 2003) or by inhibiting a TFIIH function in the phosphorylation of the Rpb1 carboxyl-terminal domain (Schang et al. 2000). Sub1 also influences the phosphorylation states of Rpb1 carboxyl-terminal domain (Calvo & Manley 2005). Furthermore, Sub1 genetically interacts with Kin28, a subunit of TFIIH with kinase activity (Calvo & Manley 2005). Whether these PC4-like functions relate to the transcription mechanisms in IMD2 gene regulation is not known.
In the present study, we focused on the repressive function of Sub1. Together with the previous findings, Sub1 seems to have two modes for gene regulation, repression and activation. The fact that Sub1 is highly localized on the IMD2 gene under the induction condition may reflect its activation function, even in IMD2 gene regulation. If so, there would be a regulatory mechanism to switch between these two modes. One possible mechanism is that TFIIH is involved in this process, as the repressive mode of PC4 is alleviated by TFIIH in vitro (Fukuda et al. 2003). Another possible mechanism is that Sub1 phosphorylation state affects this process, because the phosphorylation state modulates many Sub1 and PC4 biochemical activities (Henry et al. 1996; Werten et al. 1998; Schang et al. 2000). Examining the roles of Sub1 in switching between the non-induction and the induction states of IMD2 gene expression might be informative.
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Experimental procedures |
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Table 1 summarize the yeast strains used in this study. The DST1 null mutant strain, HKY01, was derived from YPH499; the DST1 gene was disrupted using a dst1::URA3 targeting vector (Ubukata et al. 2002; Koyama et al. 2003). Gene disruption of the SUB1 gene was carried out using a sub1::HIS3 targeting vector. Vector structure and gene disruption procedures for the SUB1 gene were as previously described (Koyama et al. 2007b). The rpb9 is a gift from Dr T. Nakanishi.
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Plasmids containing mutated SUB1 genes were constructed as follows. A fragment encoding both the wild-type SUB1 gene promoter and the open reading frame (ORF) was amplified by polymerase chain reaction (PCR) using yeast genomic DNA as a template with a pair of primers: 5'-ACGTATCTCGAGCTC GTCAATG-3', located 0.6 kb upstream of the translation initiator ATG codon of the SUB1 gene, and 5'-ACCCGGTACCATTC TCACTGTG-3', located at the TGA stop codon (the underlined sequence on the complementary strand) of the SUB1 gene. The amplified fragment was cloned at the SmaI site of the pYO324 plasmid (Ohya et al. 1991). The resulting wild-type SUB1-encoding plasmid was then used as a PCR template to construct mutated SUB1-encoding plasmids. The primers used were 5'-TAATAGT CCTGGAAGTTGAGC-3' and 5'-GTCGGACAATAGTCTAACCA-3' for del-1, 5'-TCTGGACTATCCGCATCAGA-3' and 5'-CATAGCGCTTGTGTTTGTAT-3' for del-2, 5'-GTCGGACA ATAGTCTAACCA-3' and 5'-AGGTGGAGCAGGTTTCTTCT-3' for del-3. The resulting PCR products were self-ligated to generate plasmids as shown in Fig. 3A. C-terminal FLAG-tagged Sub1 protein expression vector was constructed by insertion of FLAG sequences into the wild-type SUB1-encoding plasmid.
A plasmid harboring the IMD2 gene (pYIMD2) was constructed as follows. A fragment containing the IMD2 gene promoter and an ORF was amplified by PCR using yeast genomic DNA as template with a pair of primers: 5'-CAGAGCGGATGTTGTAGGAT-3', located 0.54 kb upstream of the translation initiator ATG codon of the IMD2 gene, and 5'-GGACATACGGACAGCACTAA-3', located 0.07 kb downstream of the TGA stop codon of the IMD2 gene. The amplified fragment was cloned at the SmaI site of the pYO323 plasmid.
The reporter plasmid harboring the IMD2 gene promoter fused to the lacZ gene (Reporter #1) was constructed as described below. The corresponding region (–436 to +106) of the IMD2 gene was amplified by PCR using pYIMD2 as the template with a pair of primers 5'-TGGCCATGGCAGAGCCGGATGTTGTAGGAT-3' and 5'-TGCGGATCCTGCTTTTGCTACTTGTGGAG-3', resulting in fragments carrying a NcoI site and a BamHI site at each end (the underlined sequence or the double-underlined sequence, respectively). The fragment was digested with NcoI and BamHI, then inserted into the plasmid backbone (also digested with NcoI and BamHI,) prepared as described below. The plasmid backbone was derived from pWLac (Koyama et al. 2003), which harbors the lacZ ORF downstream of the ADH1 promoter of pACT2 (BD Biosciences, Lexington, KY). PCR using pWLac as template with a pair of primers 5'-TATCCATGGAGACGGTCACAGCTTGT CTG-3' and 5'-TCCGGATCCATGACCATGATTACGGAT TCA-3' was carried out to obtain a fragment containing the lacZ ORF but lacking the ADH1 promoter region. The resulting backbone fragment also carried an NcoI site and a BamHI site at each end (the underlined sequence or the double-underlined sequence, respectively). Reporters #2–5 were constructed by PCR using Reporter #1 as a template with pairs of primers to introduce mutations or deletion in the IMD2 gene promoter region as described below, and then self-ligating the resulting PCR products, respectively. Reporter #2 harbors the IMD2 gene promoter lacking –36 to +106, resulting in the deletion of the RE including the TATA2 box. Reporter #3 harbors a TATA2 mutated to TGCAGATTTT. Reporter #4 harbors the IMD2 gene promoter lacking –175 to –37, resulting in the deletion of the intermediate region between the GRE and the RE. Reporter #5 harbors a GRE mutated to TACCCATA.
Drug sensitivity assays
Yeast strains were cultivated at 30 °C in SD medium supplemented with appropriate nutrients until the optical density at 600 nm (OD600) reached approximately 2.8 (early stationary phase). The cultures were serially diluted sixfold, and 10 µL of each diluted culture was spotted on to SD agar plates containing MPA or no drug. Glucose was used as sole carbon source in SD agar plates. The plates were incubated at 30 °C for 3–4 days.
Northern blot analyses and short transcripts assay
Yeast strains were cultivated at 30 °C in SD medium supplemented with the appropriate nutrients until the OD600 reached approximately 0.5 (mid-log phase), and MPA was then added. After incubation with 15 µg/mL MPA for 0, 1 and 4 h, cells were collected by centrifugation, and total RNA was extracted. For Northern blot analysis, total RNA (10 µg) was loaded onto each lane of an agarose gel containing formaldehyde. The probes were prepared from PCR products amplified from pYIMD2 with 5'-GTGGTATGTTGGCCGGTACTACC-3', located 0.4 kb upstream from the TGA stop codon of the IMD2 gene, and 5'-TCAGTTA TGTAAACGCTTTTCGTA-3', located at the TGA stop codon (underlined sequence) of the IMD2 gene, or from the PCR products amplified from yeast genomic DNA with the primers described previously for the short transcripts assay (Davis & Ares 2006), and then labeled with [
-32P]dCTP using the random priming method. The probes were hybridized with the blots at 55 °C for 1 day. The blots were washed with 2 x saline sodium citrate (SSC) containing 0.1% SDS and with 0.1 x SSC containing 0.1% SDS with agitation at room temperature, and then visualized by autoradiography.
Nucleotide pool size analyses
Yeast strains were cultivated at 30 °C in SD medium containing 10 µCi/mL [32P]orthophosphate until the OD600 reached approximately 0.1, and then MPA was added. After incubation under 0.2 µg/mL MPA for 0 and 0.5 h, the cultures were passed through nitrocellulose membrane filters. The filters were subjected to extraction with 1 M formic acid saturated with 1-butanol (Kawamura et al. 2005). The extracts were subjected to thin layer chromatography using polyethyleneimine-cellulose plates as described previously (Kawamura et al. 2005). The plates were subjected to autoradiograph, and the spots corresponding to GTP and ATP were quantified.
β-Galactosidase assays
β-Galactosidase activity was assayed as described previously (Koyama et al. 2007a), except that yeast strains were cultivated at 30 °C in SD medium supplemented with the appropriate nutrients until the OD600 reached approximately 0.1, and then MPA to give 0.03 µg/mL was added. After 6-h incubation with MPA, cells were collected by centrifugation. The cells were lysed essentially as described previously (Koyama et al. 2007b) except that the rigorous mixing with acid-washed glass beads were carried out with three cycles of 5-min rigorous mixing at 4 °C followed by a 5-min incubation on ice. β-Galactosidase activity was assayed as described previously (Koyama et al. 2007b).
Chromatin immunoprecipitation (ChIP) analyses
ChIPs were carried out essentially as described previously (Calvo & Manley 2005). Briefly, wild-type yeast strain harboring either the Sub1-FLAG expression vector or the FLAG expression vector were grown to OD600 approximately 0.8 in SD medium at 30 °C. Formaldehyde was added to a final concentration of 1%, and the mixture was incubated for 20 min. Cells were lysed with glass beads in FA lysis buffer (50 mM Hepes–KOH at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail, Sigma Chemical Co., St Louis, MO). Chromatin was sheared by sonication, so that the average fragment size was between 200 and 500 base pairs (determined by agarose gel electrophoresis). We used anti-FLAG antibody (Sigma Chemical Co.) to immunoprecipitate FLAG-tagged Sub1. For immunoprecipitations, antibodies were incubated with chromatin solution, and protein A-Sepharose CL-4B beads (Amersham/GE Healthcare, Piscataway, NJ) were added. To quantify immunoprecipitated DNA, real-time PCR was carried out on an ABI Prism 7700 Sequence Detection System using SYBR Premix ExTaq (Takara Bio, Otsu, Japan). Values of immunoprecipitated PCR products were compared to those of the total input. The sequences of primer sets were as follows (sense primer/antisense primer): IMD2 promoter: 5'-TTGGTAAAAATTTCGGCTGGA-3'/5'-AGCCACCAAGATT CTCCGG-3'; IMD2 ORF: 5'-CGCCATGCAAAAGACTGGTA-3'/5'-GTCAACGACAGCACCGGAG-3'; ACT1 promoter: 5'-TCTTCTTTACCCGCCACGC-3'/5'-TGCAAGAGGACGTG GAAGAAA-3'; the non-transcribed HMR gene: 5'-CCAACA TTTTCGTATATGGCGATA-3'/5'-TCTTGTGCAAATTCCAA CTAAAGG-3'.
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Acknowledgements |
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strain, and Dr Karin Mesches and Dr Michael H. Mesches for proofreading of the manuscript. This work was supported by a research grant from Japan Society for the Promotion of Science (JSPS) to T.I. H.K. and M.N. are recipients of the postdoctoral JSPS Research Fellowship for Young Scientist. |
Footnotes |
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* Correspondence: sekimizu{at}mol.f.u-tokyo.ac.jp
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Accepted: 4 August 2008
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