Introduction

High-gravity fermentation is applied in the brewing industry to enhance the brewing capacity by diluting the produced beers with higher alcohol content to beers with the desired alcohol content (about 5%) (Almeida et al. 2001; Reilly et al. 2004). Due to the constantly changing environmental conditions during these industrial fermentations, the yeast performance is negatively affected. Consequently, utilization of the fermentable sugars in wort is often incomplete resulting in aberrant flavour profiles and a lower yield of ethanol (Rautio et al. 2007; Dietvorst et al. 2005; Rautio and Londesborough 2003). Two sets of genes, the FLO and the MAL genes, are important during high-gravity fermentation and these are located at the subtelomeric regions. The FLO genes are important for the sedimentation of yeast after fermentation to improve product clarification (Govender et al. 2008; Verstrepen and Klis 2006; Teunissen and Steensma 1995) while the MAL genes are important for the utilization of maltose during fermentation (Dietvorst et al. 2007; Vidgren et al. 2005; Kodama et al. 1995). Premature flocculation negatively affects efficient utilization of fermentable sugars and co-regulation of MAL and FLO can thus be detrimental to optimal fermentation performance. In the budding yeast Saccharomyces cerevisiae, telomeric regions as well as the HML/HMR silent mating-type loci and the ribosomal DNA (rDNA) repeats are subjected to silencing. Silencing is a gene-independent form of repressed chromatin structure that affects specific domains of the yeast genome. Regions of repressed chromatin are often hypoacetylated, whereas actively transcribed chromosomal regions contain hyperacetylated histones (reviewed in Perrod and Gasser 2003). Complex proteins associated with Set1 (COMPASS) mediated silencing of the expression of the subtelomerically located FLO and MAL genes occurs during high-gravity fermentation (Houghton-Larsen and Brandt 2006; Dietvorst and Brandt 2008). In the present study, we investigated whether the absence of other histone modifying proteins than the subunits of COMPASS, that have been reported to be involved in (sub)telomeric silencing, also affected the performance of yeast during high-gravity fermentation.

The silent information regulator (SIR) complex, which comprises the histone modifying Sir2, Sir3 and Sir4 proteins, is required for repressing transcription at the HMR and HML silent mating type loci (Rine and Herskowitz 1987) and at the telomeres (Gasser and Cockell 2001). The SIR complex is recruited to the chromosome via interaction with specific DNA sequences termed silencers. Telomeric silencers are composed of an array of Rap1 binding sites embedded in the telomeric (TG1–3) n repeats (reviewed in Rusche et al. 2003). Once recruited to the telomeric silencer, a complex formed by Sir2, Sir3 and Sir4, involving yKU70/80 for its formation, spreads along the chromosome via interaction with histone tails (Luo et al. 2002).

Histone deacetylases (HDACs) are transcriptional repressors that reduce acetylation of histones to create regions of repressed chromatin. In addition to SIR2, the yeast genes RPD3, HDA1 and HST2 are also known to encode HDACs in S. cerevisiae. HDACs Rpd3 and Hda1 share significant sequence similarity but are found in two different complexes involved in transcriptional silencing (Rundlett et al. 1996). Hda1, Hda2 and Hda3 are subunits of a histone deacetylase (HDA) complex that is structurally distinct from the complex formed by the Rpd3, Sin3 and Sap30 proteins (Rundlett et al. 1996; Sun and Hampsey 1999; Wu et al. 2001). Disruption of HDA1 or RPD3 increases acetylation of histone H3 and H4 resulting in fewer cells expressing the URA3 reporter gene integrated at the telomeric end of chromosome V R (Rundlett et al. 1996).

Many other histone modification enzymes such as Hat1, Gcn5 and Dot1 have been shown to be involved in silencing. The Hat1 protein is the catalytic subunit of the histone acetyltransferase complex Hat1–Hat2 that is involved in telomeric silencing and DNA double-strand break repair (Kelly et al. 2000; Qin and Parthun 2002). Significant silencing was observed when deletion of HAT1 or HAT2 was combined with mutations of specific lysine residues in the histone H3 NH2-terminal tail, while there was no change in the level of silencing on a telomeric gene in the absence of HAT1 or HAT2 (Kelly et al. 2000). Another histone acetyltransferase (HAT), Gcn5, is the catalytic subunit of three chromatin-modifying complexes, Spt–Ada–Gcn5 acetyltransferase (SAGA), ADA and SLIK/SALSA. It has been shown that numerous genes are transcriptionally regulated by these complexes (Eberharter et al. 1999; Sterner et al. 2002; Pray-Grant et al. 2002). Strikingly, deletion of GCN5 is reported to enhance silencing of the URA3 reporter gene integrated at the telomeric end of chromosome V R (Sun and Hampsey 1999). The Dot1 protein was isolated as a high copy disruptor of telomeric silencing and has been linked to methylation of histone H3 lysine 79 (van Leeuwen et al. 2002; Ng et al. 2002; Lacoste et al. 2002). In the absence of the Dot1 protein, silencing of telomeres is reduced (Singer et al. 1998). Moreover, mislocalization of silencing regulators Sir2 and Sir3 have been reported to be a result of deletion of DOT1 (San-Segundo and Roeder 2000).

In this study, the influence of the histone modifying proteins introduced above on flocculation and maltose consumption during high-gravity fermentation were analysed in three different maltose fermenting yeast strains. Strain 192 was used for studying flocculation behaviour under high-gravity fermentation conditions since in this strain, absence of the COMPASS components Spp1, Swd3 or Set1 induces flocculation after approximately 50 h of maltose fermentation (Dietvorst and Brandt 2008). Strain 99R displays flocculation even in media containing low concentrations of sugar and was therefore used to study flocculation induced by 10% glucose or 5% ethanol (Dietvorst and Brandt 2008). Strain Y55a was used for analysing maltose fermentation since absence of COMPASS significantly improved the capacity of strain Y55a to ferment high concentrations of maltose (Houghton-Larsen and Brandt 2006). We now report that HDAC Hda1 as well as HAT Gcn5 plays a role in regulating expression of the FLO genes preventing flocculation under high-gravity fermentation conditions. The absence of HAT Gcn5 also improved utilization of high concentrations of maltose.

Materials and methods

Yeast strains

The yeast strains used in this study are listed in Table 1. Those yeast strains that contain a single MAL1 locus are based on the prototrophic Saccharomyces cerevisiae Y55 (McCusker and Haber 1988) derivative JT20149 (MATa MAL1) that was obtained from Prof. J. M. Thevelein (KU Leuven, Belgium) and will be referred to as strain Y55a. Strain Y55a is a non-flocculent strain. Strain 192 contains a single MAL4 locus and is only flocculent in the absence of COMPASS. Strain 99R is a maltose fermenting and flocculent strain. The kanMX-cassette was removed from strain M5675 by the lox-out procedure with Cre recombinase (Houghton-Larsen and Brandt 2006), yielding strain M5758. A PCR-based strategy (Wach et al. 1994) was used to delete SIR2, SIR3, SIR4, HAT1, DOT1, GCN5, RPD3, HDA1 or HST2 from the haploid yeast strains Y55a and 192. In addition, SIR2, HDA1 or RPD3 were deleted from the haploid yeast strain 99R. The same PCR-based strategy was used to delete GCN5 from yeast strain M5758 and SWD3 from yeast strains M5993 and M6021. Standard methods were used for yeast transformation (Gietz et al. 1995). Chromosomal DNA constituting the open reading frames of these genes was replaced by the kanMX-cassette flanked by two loxP sites or by the nat1-gene. The kanMX-cassette was amplified from plasmid pUG6 (Gueldener et al. 2002) and the nat1-gene from plasmid pAG36 (Goldstein and McCusker 1999). The oligonucleotides used for amplification of these fragments contain sequences that allow integration by homologous recombination and are listed in Table 2. All deletion strains were checked by PCR analysis as described previously (Dietvorst and Brandt 2008).

Table 1 Saccharomycescerevisiae strains used in this study
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Table 2 Oligonucleotides
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Media

Yeast cells were cultivated for 24 h in YPD (1% yeast extract, 2% peptone, 2% glucose) at 30°C. When required, 300 μg/ml G418 or 150 μg/ml nourseothricin was added. Fermentations were carried out with complex medium, containing 1% yeast extract, 2% peptone and one of the sugars maltose or glucose as described previously (Houghton-Larsen and Brandt 2006).

Fermentations

Stirred miniaerobic fermentations, containing 200 ml medium, were performed at 22°C as described previously (Dietvorst and Brandt 2008). Densities of the fermentation media were determined with a Density Meter (Anton Paar DMA35n) and expressed in degrees Plato (°P) corresponding to the percentage of sucrose by weight.

Stirred microaerobic fermentations at room temperature, containing 4 ml medium, were inoculated with 60 μl from a pre-culture grown aerobically for 72 h in YPD at 30°C.

Flocculation assays

Fermentation cultures were sampled from the middle of the tubes at the beginning and at the end of fermentation. Samples were used directly for analysis or stored on ice for maximum 1 h. Flocculation was quantified by following the absorbance during floc settling in a yeast suspension after agitation as described previously (Dietvorst and Brandt 2008). The extent of flocculation was calculated by the following formula: Flocculation (%) = 100 × (A 600(control) − A 600(sample))/A 600(control).

Semi-quantitative RT-PCR

Cells were harvested from 4 ml stirred fermentations performed in complex medium containing 20% maltose at room temperature. RNA was prepared as described previously (Houghton-Larsen and Brandt 2006) and RNA samples were treated with RNase-free DNase (USB). RT-PCR was performed using the FideliTag RT-PCR Master mix (USB) according to the manufactures instructions. Briefly, 50 ng of DNase treated RNA and 10 μM of forward and reverse primers (Table 2) were incubated at 42°C for 30 min with the FideliTag mix containing reverse transcriptase. The resulting cDNA was linearly PCR amplified for 20 cycles of 30 s at 95°C, 30 s at 55°C and 2 min at 68°C. PCR products were analysed on 1% agarose gel in 1× TAE buffer. ACT1 transcript for actin was used as a control. Standard PCR did not amplify DNA fragments using similar RNA preparations as templates.

Results

Influence of histone deacetylases Hda1, Rpd3 and Sir2 on flocculation and fermentation during high-gravity maltose fermentation

Methylation of lysine 4 of histone H3 by COMPASS leads to subtelomeric silencing, as does deacetylation of lysine residues in the tail of histone H3 or H4 by HDACs Hda1, Rpd3 or Sir2 in S. cerevisiae (Rundlett et al. 1996; Krogan et al. 2002; Rusche et al. 2003; Mueller et al. 2006). Therefore, we asked if deletion of HDA1, RPD3 or SIR2 also could affect the flocculation behaviour of strain 192 or the maltose fermentation capacity of strain Y55a.

HDACs Hda1 and Rpd3 are encoded by the highly similar HDA1 and RPD3 genes (Rundlett et al. 1996) but only Hda1 prevented flocculation in strain 192 during high-gravity conditions. After 50 h of fermentation, large aggregates of cells were present in cultures of strain 192 deleted for SWD3 and after approximately 70 h, cultures of strain 192 deleted for HDA1 displayed a similar pronounced flocculation (Fig. 1a). Cells with both SWD3 and HDA1 deleted, showed a similar flocculation profile as strain 192 deleted for SWD3 only (Table 3). Semi-quantitative RT-PCR analyses suggested that increased expression of FLO1 in the hda1 mutant, like in the swd3 mutant, caused the flocculation (Fig. 1b). Interestingly, the expression of FLO9 was low in the hda1 mutant, while the expression of both FLO1 and FLO9 was elevated in the swd3 mutant (Fig. 1b). The swd3 hda1 double mutant showed a similar expression level for FLO1 and FLO9 as the swd3 mutant (data not shown). The low expression of FLO9 in the hda1 mutant may indicate that Hda1 exerts a locus specific effect on FLO1 expression.

Fig. 1
figure 1

Flocculation of the mutant strain 192 deleted for HDA1 during high-gravity maltose fermentation. After 72 h fermentation of complex medium containing 21.8°P maltose, the flocculation phenotype was photographed for the wild-type strain 192 (WT) and strain 192 deleted for HDA1 (M5993) or SWD3 (M5845). Photographs were taken immediately after switching off the stirring-device and at 20 s intervals. The time in seconds is indicated under each photograph (a). Primers specific for the yeast genes FLO1, FLO9 and ACT1 were used for semi-quantitative RT–PCR amplification using 50 ng total RNA extracted from wild-type strain 192 and strain 192 deleted for SWD3 or HDA1 (b)

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Table 3 Overview of different phenotypes analysed in this study
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Strain Y55a deleted for HDA1 showed a similar fermentation profile as the wild-type strain Y55a (Table 3). The mutant strain deleted for RPD3 fermented complex medium with 22.2°P maltose less complete (about 1.5°P) than the wild-type strain Y55a (Table 3) indicating that Rpd3 function to some extent influences utilisation of maltose. These results are in line with previous findings that HDACs are transcriptional repressors and deletion of RPD3 resulted in an overall increase in the acetylation of histones H3 and H4 (Zhou et al. 2009) and an increase in silencing of reporter genes inserted near telomeres (Rundlett et al. 1996; Sun and Hampsey 1999).

Flocculation was never detected in strain 192 deleted for SIR2 during the high-gravity maltose fermentations. At the end of fermentation, flocs consisting of many thousands of cells were present in the swd3 mutant, while both wild-type and sir2 cells only formed micro-clumps of about 10–20 cells (Fig. 2a). Flocculation was also quantified using a photometric assay (Fig. 2b). The absence of a functional COMPASS resulted in a strong flocculation phenotype at the end of fermentation, while flocculation was barely detectable in the wild-type strain or the sir2 mutant (Fig. 2b). Semi-quantitative RT-PCR analyses showed elevated expression levels of FLO1 and FLO9 in the swd3 mutant while expression levels in the sir2 mutant were similar as in the wild-type strain 192 (Fig. 2c).

Fig. 2
figure 2

Lack of the silent informator regulator complex component Sir2 has no effect on flocculation of strain 192 during high-gravity maltose fermentation. Fermentation of complex medium with 20.8°P maltose with wild-type strain 192 (WT) and strain 192 deleted for SWD3 (M5845) or SIR2 (M5853) was carried out in duplicate, and the error bars indicate the range between duplicate experiments. Microscopy was performed on cells harvested at the end of fermentation to visualise floc formation in the wild-type strain 192 and strain 192 deleted for SWD3 or SIR2 (a). Flocculation was quantified by measuring the residual absorbance of the yeast suspension after agitation and subsequent undisturbed standing for 15 min (see “Materials and methods”). Relative flocculation levels were determined at the start and at the end of fermentation (b). Primers specific for the yeast genes FLO1, FLO9 and ACT1 were used for semi-quantitative RT–PCR amplification using 50 ng total RNA extracted from wild-type strain 192 and strain 192 deleted for SWD3 or SIR2 (c)

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Strain Y55a deleted for SIR2 displayed both slightly enhanced and impaired high-gravity maltose fermentations (Table 3). More precisely, in two fermentation experiments the sir2 mutant fermented maltose faster and more complete compared to wild-type Y55a while a slower and less complete fermentation of the sir2 mutant was observed in four experiments. In addition, the SIR2-deleted Y55a strain often showed variation in the duplicates of stirred fermentations. Together, this suggests that the ability to ferment high concentrations of maltose is inconsistent in cells deleted for SIR2. Absence of the Sir2 protein has been reported to result in decreased chromosome stability and decreased lifespan (Holmes et al. 1997), which might explain the observed variations in fermentation capacity. Strain 192 deleted for SIR3, SIR4, HST2 (homologue of Sir2) or DOT1 (Disruptor of Telomeric silencing) did not flocculate during high-gravity fermentation nor did the deletion of these genes improve maltose fermentation capacity in strain Y55a (Table 3).

Previously, we reported that S. cerevisiae maltose-fermenting strain 99R displays flocculation when the glucose concentration in the growth medium is increased to 10%. Strain 99R deleted for SWD3 even displayed a Ca2+-dependent flocculation after growth in YP containing 2% glucose for 18 h at 30°C (Dietvorst and Brandt 2008). The characteristics of strain 99R were used to analyse the effect of deleting the HDACs Hda1, Rpd3 or Sir2 on flocculation induced by 10% glucose or 5% ethanol. Flocculation was induced in wild-type 99R cells when grown in 10% glucose or 2% glucose with 5% ethanol (Fig. 3). Cells deleted for HDA1 or RPD3 displayed similar flocculation behaviour as the wild-type while cells deleted for SIR2 failed to flocculate (Fig. 3). This result suggests that the flocculation of strain 99R induced by high glucose concentration or ethanol, is dependent on the Sir2 protein.

Fig. 3
figure 3

Flocculation induced by high glucose concentration or ethanol is Sir2 dependent in strain 99R. Cells of wild-type strain 99R (WT) and strain 99R deleted for SWD3 (M6013), HDA1 (M6025), SIR2 (M6014) or RPD3 (M6024) were grown for 18 h at 30°C in 4 ml complex medium containing 2% glucose (2%D), 2% glucose with ethanol (EtOH) or 10% glucose (10%D). Flocculation was photographed after allowing the yeast to settle at room temperature for 5 min

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Influence of histone acetyl transferases Gcn5 and Hat1 on flocculation and fermentation capacity during high-gravity maltose fermentation

The steady state level of acetylation of histones is a balance between the action of the HDACs and the histone acetyltransferases (HATs). Parallel to the HDACs described in the previous section, the effect of the absence of HATs Gcn5 and Hat1 on the flocculation behaviour of strain 192 or the maltose fermentation capacity of strain Y55a during high-gravity maltose fermentation was investigated.

Cultures of strain 192 deleted for GCN5 displayed pronounced flocculation after approximately 70 h of fermentation (Fig. 4a). In addition, strain 192 deleted for both SWD3 and GCN5 showed similar flocculation as strain 192 deleted for SWD3 and HDA1 or SWD3 alone (Table 3). Semi-quantitative RT-PCR analyses showed that the expression of both FLO1 and FLO9 was elevated in the gcn5 mutant compared to the levels in the wild-type strain at time points when flocculation was visible (Fig. 4b).

Fig. 4
figure 4

Flocculation phenotype of the gcn5 mutant in strain 192 during high-gravity maltose fermentation. After 72 h fermentation of complex medium containing 21.8°P maltose, the flocculation phenotype was photographed for strain 192 deleted for GCN5 (M6021). Photographs were taken immediately after switching off the stirring-device and at 20 s intervals. The time in seconds is indicated under each photograph (a). Primers specific for the yeast genes FLO1, FLO9 and ACT1 were used for semi-quantitative RT–PCR amplification using 50 ng total RNA extracted from wild-type strain 192 (WT) and strain 192 deleted for SWD3 (M5845) or GCN5 (M6021) (b)

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Strain Y55a lacking Gcn5 fermented high concentrations of maltose more complete (about 2.5°P) than the wild-type (Fig. 5a) while Y55a deleted for both GCN5 and SWD3 fermented high concentrations of maltose less complete (about 1.5 P) than the wild-type (Fig. 5b). However, deletion of both SWD3 and GCN5 resulted in severe defects in cell growth. Growth in YPD liquid medium of strain Y55a deleted for GCN5 was slower than the wild-type strain (doubling times were 87 ± 5 min and 74 ± 1 min, respectively) while the doubling time was even longer (103 ± 2 min) for strain Y55a deleted for both SWD3 and GCN5. This is in line with previously published observations (Zhang et al. 1998). From these analyses, it is not possible to determine if the reduced completeness and slower speed of high-gravity maltose fermentation resulted from these defects in cell growth or that Gcn5 is directly regulating maltose utilisation (Fig. 5b).

Fig. 5
figure 5

Histone acetyltransferase Gcn5 affects the fermentation capacity of strain Y55a during high-gravity maltose fermentation. Fermentation of complex medium with 22°P (a, b) maltose with the wild-type strain Y55a (WT) and strain Y55a deleted for GCN5 (M6019) (a) or deleted for SWD3 as well as GCN5 (M6037) (b). Fermentations were done in duplicate, and the error bars indicate the range between duplicate experiments

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Deletion of HAT1 in strain 192 did not affect flocculation and strain Y55a lacking Hat1 fermented high-gravity maltose similar as the wild-type (Table 3), indicating that the role of the histone modifying Hat1 protein is insignificant during high-gravity maltose fermentation. However, lack of Hat1 improved high-gravity fermentation performance in S. cerevisiae strain 192 (Table 3). COMPASS-mediated improvement of fermentation of high concentrations of maltose in strain 192 was previously found to be insignificant, possibly hidden by the heavy premature flocculation of the COMPASS mutants (Dietvorst and Brandt 2008). The other histone modifying proteins analysed in this study did not affect high-gravity maltose fermentation of strain 192 (Table 3). Together this indicates a strain specific role for the Hat1 protein in strain 192 during high-gravity fermentation, as was previously found for COMPASS-mediated silencing of the MAL genes that also appears to be strain-dependent (Dietvorst and Brandt 2008).

Discussion

Efficient beer fermentation requires the rapid and complete utilisation of the main fermentable sugars in wort. Due to decreased yeast performance because of environmental stresses imposed during high-gravity conditions, fermentations of complex medium containing high concentrations of maltose are often incomplete. The subtelomerically located FLO and MAL genes are important for the performance of yeast during these fermentations. An important control on the expression of these genes is COMPASS-mediated silencing of the FLO and the MAL genes. Absence of COMPASS mediated silencing leads to induced flocculation and improved maltose fermentation, respectively (Dietvorst and Brandt 2008; Houghton-Larsen and Brandt 2006).

In the present work, we analysed the effect of several other genes encoding histone-modifying proteins on flocculation and fermentation capacity (Table 3). We show in addition to COMPASS that the Hda1 and Gcn5 proteins are required to repress flocculation during high-gravity fermentation (Figs. 1a, 4a). Expression of FLO1 only is induced in the absence of Hda1 (Fig. 1b) whereas the expression of both FLO1 and FLO9 is induced in the absence of Gcn5 (Fig. 4b). Whether these two histone-modifying proteins directly affect the transcription of the FLO genes is not known. However, Hda1 affects acetylation at a distance of 10–25 kb from the telomeres (Robyr et al. 2002), while Gcn5 as the catalytic subunit of three chromatin-modifying complexes, regulates transcription of numerous genes (Sterner et al. 2002; Pray-Grant et al. 2002). Subtelomeric regions affected by Hda1 appear to be continuous and both the MAL and the FLO genes are located in domains affected by Hda1 (Robyr et al. 2002). In addition to the effect on flocculation under high-gravity conditions, the absence of Gcn5 also improved efficient high-gravity maltose utilisation of strain Y55a (Fig. 5a) while Hda1 had no effect on maltose consumption. Gcn5 is required for the repression of ARG1 in rich medium (Ricci et al. 2002). These data together indicate that Gcn5 possibly affects expression of multiple genes located over regions extending 20 kb from the telomeres whereas Hda1 possibly is involved in gene specific regulation. The Flo1 protein in particular is thought to be involved in the formation of large aggregates of cells (Verstrepen and Klis 2006) and can thus alone be responsible for the strong flocculation phenotype observed during high-gravity maltose fermentations (Figs. 1, 4).

Gcn5 is a conserved histone acetyltransferase that functions as a catalytic subunit of multiple HAT complexes that primary acetylates lysine 9, 14, 18, 23, 27 and 36 of histone H3 but also lysine 11 and 16 of histone H2B and lysine 8 and 16 of histone H4 (Kuo et al. 1996; Grant et al. 1999; Suka et al. 2001; Morris et al. 2007). Both acetylation and methylation of histone lysine residues have been shown to play key roles in the regulation of chromatin structure and function, with the majority of these modifications occurring on histone H3 (Felsenfeld and Groudine 2003). The involvement of COMPASS (Dietvorst and Brandt 2008) and Gcn5 in silencing of the FLO and the MAL genes during high-gravity conditions may then be explained by a more specific role for histone H3 in silencing of genes located approximately 20 kb or more from the telomeres. Alternatively, a functional connection between histone acetylation and methylation as reported recently (Kuo et al. 2009), could also explain our data. Enhanced methylation of arginine 3 (R3) of histone H3 as a result of acetylation at any of the four acetylatable lysine residues of histone H4 by Gcn5 has been reported (Kuo et al. 2009). Thus, acetylation of histone H3 could supposedly result in more efficient methylation. In this hypothetic situation, deletion of GCN5 will reduce acetylation of histone H3 that consequently results in reduced K4H3 methylation by COMPASS, hence derepression of the FLO and the MAL genes.

Heterochromatin-like regions, formed by the actions of the Sir proteins, are also present in subtelomeric regions. However, there is virtually no overlap between these regions affected by Sir2 and the domains affected by Hda1 (Wyrick et al. 1999; Bernstein et al. 2000; Robyr et al. 2002). In absence of the Sir2, Sir3 or Sir4 proteins, strain Y55a was only marginally affected in its ability to ferment high concentrations of maltose (Table 3) and the absence of Sir function did not induce flocculation nor induce the expression of FLO1 and FLO9 (Fig. 2). Since gene silencing at the telomeres requires the silencing proteins Sir2, Sir3 and Sir4 (Gasser and Cockell 2001) and complete loss of transcriptional silencing due to absence of deacetylase Sir2 has been reported (Smith et al. 2000; Cuperus et al. 2000), a more dramatic effect of deleting SIR2 in our study was anticipated. Possibly other HDACs maintain telomeric regions in their hypoacetylated state. Since it has been reported that global histone acetylation levels were unchanged when all five members of the SIR2 gene family (SIR2 and HST1-4) were disrupted (Smith et al. 2000), it is even more likely that the hypoacetylated histones, located approximately 20 kb from the chromosome ends, have been shielded from the action of acetylases. Since FLO1 and FLO9 are located 25 up to 40 kb away from the telomere ends and MAL11, MAL12 and MAL13 are located 10 up to 20 kb away, respectively, a SIR-independent form of silencing in regions stretching further away from the telomere end could be likely. That Sir possibly indirectly may influence the expression of genes located that far from the telomere as the FLO genes was underlined by the finding that flocculation induced by 2% glucose plus 5% ethanol or by 10% glucose in strain 99R was dependent on the presence of Sir2 (Fig. 3).

This study identified the Hda1 and Gcn5 proteins, in addition to COMPASS, as being important for regulating the FLO and the MAL genes located at a distance from the telomere. As indicated in this study caution should be exercised when analysing regulation of gene expression by histone modifying activities, in particular when analysing industrial yeast strains under high-gravity conditions. Both the complexity and the possible interplay of the different histone modifying activities are perhaps reflected in the strain specific expression of phenotypes related to maltose utilisation and flocculation. The consequences of modifying these activities appear to be highly dependent on the yeast strain background and may involve both regulated gene expression and heterochromatic silencing of genes. Thus, regulation of histone modifying activities as a tool to improve the performance of industrial yeast requires careful analyses of the particular yeast strain to prevent unpredictable phenotypic consequences and to assure that the strategy indeed will be successful in a particular industrial yeast strain.