Regulation of Gene Expression by Glucose in Saccharomyces cerevisiae: a Role for ADA2 and ADA3/NGG1

Abstract

When Saccharomyces cerevisiae cells are transferred from poor medium to fresh medium containing glucose, they rapidly increase the transcription of a large group of genes as they resume rapid growth and accelerate progress through the cell cycle. Among those genes induced by glucose is CLN3, encoding a G₁ cyclin that is thought to play a pivotal role in progression through Start. Deletion of CLN3 delays the increase in proliferation normally observed in response to glucose medium. ADA2 and ADA3/NGG1 are necessary for the rapid induction of CLN3 message levels in response to glucose. Loss of either ADA2 or ADA3/NGG1 also affects a large number of genes and inhibits the rapid global increase in transcription that occurs in response to glucose. Surprisingly, these effects are transitory, and expression of CLN3 and total poly(A)⁺ RNA appear normal when ADA2 or ADA3/NGG1 deletion mutants are examined in log-phase growth. These results indicate a role for ADA2 and ADA3/NGG1 in allowing rapid transcriptional responses to environmental signals. Consistent with the role of the Ada proteins in positive regulation of CLN3, deletion of RPD3, encoding a histone deacetylase, prevented the down regulation of CLN3 mRNA in the absence of glucose.

When growing cells of the yeast Saccharomyces cerevisiae deplete the glucose from normal YEPD growth medium, they decrease both mRNA and protein biosynthesis as they pass first through the diauxic shift from fermentative to oxidative growth on ethanol. Eventually the cells deplete a limiting nutrient and approach stationary phase (36). As this process proceeds, G₁ length steadily increases, allowing the cell cycle to maintain pace with the steadily decreasing rate of growth in mass. Cells that fail to return to rapid growth after transfer to rich medium are at a considerable selective disadvantage. All else being equal, the more rapidly a cell returns to logarithmic growth in glucose, the more descendants it will have. The rapid return to growth after transfer to fresh medium involves a large-scale induction of gene expression (36). Previous work using labeled poly(dT) probes suggested that the level of total mRNA in log-phase cells is up to 10-fold higher than in post-log-phase and stationary-phase cultures (3). More recent work with cDNA microarrays has helped to clarify this picture by showing that as cells reached glucose exhaustion at the diauxic shift, the transcription of approximately 17% of the 6,100 genes examined was decreased by glucose exhaustion (6). Therefore, the transcriptional machinery is able to recognize a large set of genes that are induced by glucose. Among the genes turned on in glucose medium are those encoding glycolytic enzymes, as well as genes promoting protein translation. The emerging picture therefore suggests that yeast cells make a coordinated response to glucose that allows them to rapidly increase their rate of growth on this rich resource. How this process is regulated remains poorly understood.

As cells return to logarithmic growth, the rates of both growth in size and progression through the cell cycle are accelerated. We have previously shown that levels of mRNA encoding Cln3, a G₁ cyclin, rapidly increase in response to fresh medium (19). More recently, we have shown that CLN3 transcription is regulated by carbon source (25) and that regulation of CLN3 expression plays a role in the regulation of G₁ length in response to nutrient changes (15, 24).

In this work, we show that the level of CLN3 mRNA increases in parallel with a large-scale increase in total poly(A)⁺ message in response to glucose. We also show that CLN3 plays a role in the rapid acceleration of growth in response to fresh medium. We show that ADA2 and ADA3/NGG1 are needed not only for the rapid increase in CLN3 message but also for the rapid global transcriptional increase that normally occurs in response to glucose. ADA2 and ADA3/NGG1 encode transcriptional adaptor proteins that form large complexes associated with histone acetyltransferase activity (1, 2, 13, 22, 26, 29). These proteins are believed to play an important role in the transcriptional regulation of a large number of genes. Surprisingly, although ADA2 and ADA3/NGG1 were important in the rapid increase in mRNAs induced by glucose, deletion of ADA2 or ADA3 had relatively little impact on the cellular levels of these mRNAs over a longer time. This suggests a role for ADA2 and ADA3/NGG1 in mediating rapid responses.

MATERIALS AND METHODS

Yeast strains and culture methods.

The S. cerevisiae strains used are listed in Table 1. Deletion of CLN3 in strain DS10 was done as described elsewhere (5) and confirmed by Southern and Northern blotting. The ada2 and ada3/ngg1 deletion mutants in the CY232 (21) background, described in reference 27, were constructed by using a hisG-URA3 cassette as described previously (22). The rpd3Δ strain (DY150) and the isogenic wild-type strain were provided by David Stillman (32). Cells were grown in YEPD medium containing 1% yeast extract, 2% Bacto Peptone, and 2% glucose at 30°C with shaking. The term “post-log cells” refers to cultures grown for 2 to 3 days in YEPD to an optical density at 660 nm (OD₆₆₀) of approximately 6 to 7.

TABLE 1.

S. cerevisiae strains used

Strain	Genotype	Reference or source
HR125	MATa leu2-3 leu2-112 ura3-52 trp1-1 his3-532 his4	16
TC41	MATa leu2-3 leu2-112 ura3-52 trp1-1 his3-532 his4 cyr1::URA3 cam	16
DS10	MATa his3-11,15 leu2-3,112 lys1 lys2 ura3-52 trp1Δ	E. A. Craig
TG3	MATa his3-11,15 leu2-3,112 lys1 lys2 ura3-52 trp1Δ cln3Δ::URA3	This study
CY232	MATa his4 leu2-3,112 ura3-52 trp1-289	C. Peterson
CY232 ada2Δ	MATa his4 leu2-3,112 ura3-52 trp1-289 ada2Δ	C. Peterson
CY232 ada3Δ	MATa his4 leu2-3,112 ura3-52 trp1-289 ada3Δ	C. Peterson
W303	MATa ade2 can1 his3 leu2 trp1 ura3-52	D. Stillman
DY150	MATa ade2 can1 his3 leu2 trp1 ura3-52 rpd3Δ::LEU2	D. Stillman

RNA preparation and blotting.

RNA isolation and blotting were done as described elsewhere (9). Yeast samples (10 to 15 OD₆₆₀ U) were collected for RNA preparation, and samples (15 μg) were run on agarose-formaldehyde gels and transferred to nylon filters. Blots were probed with a 1-kb EcoRI fragment of CLN3, a 3-kb HindIII fragment of BCK2, and a 1-kb BamHI/HindIII fragment of CDC28 or with a poly(dT) oligomer (15 to 18 nucleotides) end labeled with T4 polynucleotide kinase and [γ-³²P]ATP for total poly(A)⁺ messages. A 0.6-kb SacI fragment was used to probe for U2 RNA as a loading and transfer control. Uniform loading and transfer of the blots were confirmed by rRNA staining with ethidium bromide. Blots were analyzed with a Molecular Dynamics PhosphorImager.

RESULTS

Role of CLN3 in the yeast response to glucose.

As cells growing in YEPD pass out of log phase, they enter a slow growth oxidative phase, in which they tend to accumulate in G₁ as unbudded cells. When these cells are transferred back to fresh medium, they return to rapid proliferative growth. This is preceded by a rapid increase in the transcription of CLN3, with a 5- to 10-fold increase within 5 min (Fig. 1A).

FIG. 1.

Deletion of CLN3 delays return to proliferative growth. (A) Cells (TG3) carrying a deletion in CLN3, along with the isogenic wild-type (WT) strain (DS10) as a control, were grown for 2 days in YEPD to an OD₆₆₀ of approximately 7. Cells were centrifuged and resuspended in fresh YEPD to an OD₆₆₀ of 1, and samples were collected for Northern blotting at the indicated times (minutes) after transfer. In this and other experiments, we used U2 RNA as a loading and transfer control because the more commonly used loading standards appear to be induced when cells are treated with fresh medium. (B) Cells were treated as described above, and bud counts on triplicate samples were performed at the indicated times after transfer. At least 300 cells were counted per time point for each strain. (C) A cyr1Δ strain (TC41) was grown for 2 days to post-log phase in YEPD–1 mM cAMP to an OD₆₆₀ of 7, transferred into YEP medium lacking both glucose and cAMP, and incubated overnight. The cells were then transferred to fresh YEPD without cAMP, and samples were collected at the indicated times for Northern blotting.

The prominent role that CLN3 plays in regulating transcription of Start-specific transcripts (34, 35) suggested that the induction of CLN3 mRNA might be an important step in moving out of G₁ as cells resume rapid growth. To test this, we measured the ability of post-log cells carrying a deletion in CLN3 to begin moving through the cell cycle after transfer to fresh medium (Fig. 1B). Post-log cells were transferred to fresh YEPD, and bud emergence was scored at intervals in triplicate samples to determine how quickly the cells were able to move through Start. Compared to wild-type cells, cells lacking CLN3 were delayed by one generation, approximately 90 min, in returning to proliferative growth.

The increase in transcript levels in response to glucose is not restricted to CLN3 but coincides with an increase in the level of a large group of mRNAs that can hybridize with a labeled poly(dT) oligomer (Fig. 1A). While we cannot rule out a large-scale increase in polyadenylation in this experiment, this change in poly(A)⁺ RNA has been previously shown to correspond to a change in the overall rate of mRNA synthesis by RNA polymerase II (3). Few individual bands are observable; however, most of the hybridization occurs in the size range between 500 and 4500 nucleotides, clustering around the 1,000- to 2,000-nucleotide range. This size range is consistent with the expected size for most yeast messages, and the smeared signal is also consistent with an increase in the group of more than 1,000 messages reported to be increased by glucose (6). This indicates that CLN3 is among a large group of genes that are induced in response to fresh glucose medium.

While the increase in CLN3 transcription in response to fresh medium coincides with an increase in overall mRNA levels, CLN3 is not necessary for this increase. Deletion of CLN3 delayed the return to proliferative growth, but it did not prevent the increase in total mRNA in response to fresh medium (Fig. 1A). Furthermore, even though cln3Δ cells have a delay in Start (4, 23), they grow at a normal rate after the initial delay. Both wild-type cells and cells carrying a deletion in CLN3 grew in YEPD with a doubling time of approximately 100 min (not shown).

Addition of glucose to S. cerevisiae cells has been shown to produce a rapid increase in intracellular cyclic AMP (cAMP) levels (11), and cAMP levels fall as cells deplete the glucose in the medium (12, 31). To determine whether this increase in cAMP is needed for the increase in total mRNA in response to fresh medium, we used a strain (TC41) carrying a deletion in the gene encoding adenylate cyclase, CYR1, in which we can manipulate cAMP levels. These cyr1Δ cells cannot produce cAMP and become permanently arrested in G₁ unless cAMP is added to the medium. We found that total mRNA levels responded to glucose in the complete absence of cAMP (Fig. 1C). In this experiment, the cells were grown to post-log phase in YEPD–1 mM cAMP at an OD₆₆₀ of 7. These cells were largely unbudded (less than 3%). The cells were then transferred into YEP medium lacking both glucose and cAMP and incubated overnight to ensure that both glucose and cAMP were exhausted from the medium. The cells were then transferred into fresh YEPD without cAMP, and samples were collected at intervals for Northern blotting. Although these cells remained unbudded (less than 3%) in the absence of cAMP, they produced an increase in poly(A)⁺ RNA that was similar to that seen in the previous experiment. Therefore, the large-scale induction of mRNAs by glucose is cAMP independent, and progression through the cell cycle is not necessary for this response. We have previously shown that the glucose induction of CLN3 is also cAMP independent (25). Our results are consistent with the idea that CLN3 plays an important but not exclusive role in moving the cells out of G₁ and through Start as they resume proliferative growth in response to fresh medium. Overall, the results best fit a model in which fresh glucose medium induces the transcription of a large group of genes that are important in both the rapid resumption of growth in size and the resumption of progress through the cell cycle.

Deletion of ADA2 or ADA3 inhibits glucose induction.

The yeast ADA3/NGG1, ADA2, and GCN5 genes have been implicated in a wide variety of transcriptional responses (14, 33). The products of these genes form large complexes that interact with both the general transcriptional machinery and transactivator proteins. In addition, these complexes contain histone acetyltransferase activity, which is thought to play a role in altering chromatin structure at promoters. We found that deletion of either ADA2 or ADA3/NGG1 strongly inhibited the induction of CLN3 mRNA when fresh glucose medium was added to post-log cells (Fig. 2A). Deletion of ADA2 or ADA3/NGG1 also blocked the glucose induction of two other genes involved in passing Start, CDC28 and BCK2 (8, 10, 28).

FIG. 2.

Deletion of ADA2 or ADA3/NGG1 inhibits a global transcriptional response to glucose. (A and B) Post-log wild-type (WT; CY232) and isogenic ada2Δ and ada3Δ mutant cells, as indicated, were transferred to fresh YEPD at an OD₆₆₀ of 1, and samples were collected at the indicated times (minutes) for Northern blotting with probes for specific messages or with a poly(dT) oligomer for poly(A)⁺ RNA. Blots A and B are from separate gels using the same RNA source.

A more striking phenotype for the ada3/ngg1Δ and ada2Δ mutants is shown in Fig. 2B. Deletion of ADA3/NGG1 also inhibited the large-scale induction of transcription produced when glucose was added to post-log cells. In wild-type controls, poly(A)⁺ RNA increased approximately fivefold. In contrast, addition of medium containing fresh glucose to cells carrying a deletion in either ADA2 or ADA3/NGG1 produced little increase in poly(A)⁺ RNA. Thus, ADA3/NGG1 is required not only for the rapid glucose induction of CLN3 but also for the rapid increase in transcription of a large group of genes.

While the effect ada2 and ada3/ngg1 mutants on the rapid transcriptional response to glucose was quite striking, over a longer time course, CLN3 message and total poly(A)⁺ RNA levels slowly increased toward normal in these mutants (Fig. 3). When we examined CLN3 and poly(A)⁺ RNA levels in cells growing in log phase in glucose medium, we observed little if any difference between the mutants and wild-type cells (Fig. 4). This finding indicates that ADA2 and ADA3/NGG1 appear to be important in the kinetics rather than the magnitude of the transcriptional response to glucose.

FIG. 3.

Effect of ADA2 or ADA3/NGG1 deletion transcriptional response to glucose is transitory. Post-log wild-type (CY232) and isogenic ada2Δ (A) and ada3Δ mutant (B) cells, as indicated, were transferred to fresh YEPD at an OD₆₆₀ of 1, and samples were collected at the indicated times (minutes) for Northern blotting with probes for specific messages or with a poly(dT) oligomer for poly(A)⁺ RNA.

FIG. 4.

ADA2 or ADA3/NGG1 deletions do not affect CLN3 mRNA or poly(A)⁺ RNA levels in cells in log-phase growth. Wild-type (WT; CY232) and isogenic ada2Δ and ada3Δ mutant cells, as indicated, were grown to mid-log phase in YEPD and collected for Northern blotting as indicated.

Mutations in RPD3 increase CLN3 mRNA levels.

The RPD3 gene encodes a protein with histone deacetylase activity, potentially serving the opposite function of complexes containing Ada proteins (7, 20, 30). We found that indeed, deletion of RPD3 produced a very large increase in CLN3 message levels. In particular, loss of RPD3 prevented the decrease in CLN3 levels normally seen in post-log cells (Fig. 5). In contrast to the results with the ADA2 and ADA3/NGG1 mutations, deletion of RPD3 produced little effect on overall mRNA levels, indicating that RPD3 affects the transcription of a smaller subset of genes.

FIG. 5.

Deletion of RPD3 increases CLN3 message levels. (A) Post-log wild-type (DY150) and isogenic rpd3Δ (DY1539) cells were transferred to fresh YEPD at an OD₆₆₀ of 1, and samples were collected at the indicated times (minutes) for Northern blotting with a CLN3 probe or with a poly(dT) oligomer for poly(A)⁺ RNA. (B) Quantitation of the CLN3 signal with a Molecular Dynamics PhosphorImager, normalizing for loading by using the U2 signal. (C) Quantification of poly(A)⁺ signal.

DISCUSSION

For S. cerevisiae, the presence of a fermentable carbon source such as glucose in the medium has a profound impact on growth in mass, cell cycle progression, and gene expression. Very little is known about the mechanism that produces this response. It is tempting to speculate about the possible mechanisms that allow such a large group of genes to be activated by one signal. Glucose produces effects on a great number of genes with no obvious regulatory features in common. One possibility for coordinated regulation of these genes would be for glucose to regulate the activity of the ADA gene products. This would allow a centrally regulated complex, or set of complexes, to coordinate the activity of a large group of genes.

The discovery of histone acetyltransferase complexes containing the Ada proteins has led to models in which these complexes are recruited to promoter regions in order to alter chromatin structure and increase transcription. Exactly which genes require these complexes for activation remains unknown; however, it seems likely that this type of mechanism is involved in the transcriptional regulation of a wide range of genes. Our initial results indicate that loss of ADA2 or ADA3 affects the transcription of enough genes in yeast to produce a noticeable effect on the total level of poly(A)⁺ RNA at early time points. Addition of fresh glucose medium to post-log cells produced a rapid rise in poly(A)⁺ RNA levels that was strongly inhibited by loss of either ADA2 or ADA3. One possible interpretation of these results is that complexes containing Ada proteins are directly involved in the transcription of a substantial fraction of the genes that are upregulated by glucose. Another possibility is that these mutations affect the levels of a key regulator of transcription. In this case, the effect on induction by glucose would be indirect. Our experiments do not distinguish between these two models.

While the ADA deletions clearly had an impact on the transcriptional response to glucose, this effect was only transitory and had all but disappeared by the time that the cells reached log-phase growth. This suggests that an important role for the ADA gene products is one of catalyzing transcriptional responses that can eventually occur in their absence. Many protein-DNA interactions occur with very high affinity and consequent slow off rates. These complexes may be ill suited for rapid changes in response to regulatory signals. Complexes containing Ada proteins may play an important role in accelerating this kind of transcriptional response. Recent work with DNA microarrays has shown that loss of Gcn5, a key component of the SAGA complex that also contains Ada2 and Ada3, produces a decrease in the transcription of only about 5% of the genes in S. cerevisiae (17). However, because these experiments were done with cultures under constant growth conditions, the microarray results may lead to an underestimate of the importance of the SAGA complex in transcriptional regulation. It seems possible that while having little effect on the final transcript level, the SAGA complex may affect the kinetics of transcriptional responses for a much larger set of genes.

In our hands, the ada2Δ and ada3Δ mutants show a growth lag when inoculated into culture medium, taking noticeably longer than wild-type cells to enter logarithmic growth. This may be due to the delay in the transcriptional response to fresh medium. Other phenotypes such as slow growth, sensitivity to heat, and poor growth on minimal medium (18) may be due to longer-term transcriptional changes produced by the mutations.

The results with the rpd3Δ mutant indicate that histone deacetylation plays a major role in decreasing CLN3 transcription, consistent with the previously reported role in transcriptional repression reported for RPD3. Loss of RPD3 has been shown to increase overall levels of histone acetylation, and transcription of CUP1 and PHO5, suggesting a role in transcriptional repression. However, this simple model must be qualified by the fact that loss of RPD3 increases telomeric silencing (30). In contrast to the ADA2 and ADA3 mutations, deletion of RPD3 had little if any effect on the total poly(A)⁺ RNA in the cell, suggesting that Rpd3 regulates a smaller number of genes than the Ada proteins. This is consistent with the fact that RPD3 is a member of a family of similar proteins found in S. cerevisiae. These proteins may regulate separate subsets of genes.