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Epigenetic regulation of PGU1 transcription in Saccharomyces cerevisiae

Campbell Louw, Philip R. Young, Pierre van Rensburg, Benoit Divol
DOI: http://dx.doi.org/10.1111/j.1567-1364.2009.00599.x 158-167 First published online: 1 March 2010

Abstract

The PGU1 gene of Saccharomyces cerevisiae has been shown to encode a polygalacturonase. The polygalacturonase activity in S. cerevisiae is strain specific. There are no significant differences in the PGU1 promoter regions of strains with and without polygalacturonase activity. The PGU1 gene is subtelomeric because it is located within 25 kb of the right telomere of chromosome X. Expressions of genes located in subtelomeric regions in the yeast S. cerevisiae are inhibited compared with the rest of the genome. In this study, we showed that the deletion of genes involved in telomere silencing enhances polygalacturonase activity. PGU1 transcription and polygalacturonase activity are increased when PGU1 is shifted to a different location in the genome, away from the telomere located close to this gene, and the depletion of the histone H4 leads to an increase in PGU1 transcription. We concluded that PGU1 is silenced in strains without polygalacturonase activity due to an epigenetic effect. The results of this study suggest that PGU1 is silenced by being folded into a heterochromatin-like structure at its subtelomeric position on chromosome X. Formation of this silent structure is dependent on the Isw2p chromatin remodeling complex, its histone fold motif containing subunit Dls1p and the N-terminal tail of the H4 histone.

Keywords
  • Saccharomyces cerevisiae
  • PGU1
  • epigenetic regulation
  • telomeric silencing
  • histone

Introduction

Pectin is a generic name for a mixture of pectic substances with different compositions, but with a polygalacturonic acid backbone. It is a structural heteropolysaccharide that occurs mainly in the middle lamellae and the primary cell walls of higher plants. Pectin is degraded by a group of enzymes collectively called pectinases. These enzymes are classified according to their substrate, mode of action, action pattern and cleavage products (Jayani, 2005).

Pectolytic activity enables yeast to reach plant metabolites and thus facilitate yeast invasion of fruits (Gognies, 1999). Polygalacturonic acid is degraded by an endo-polygalacturonase encoded by the PGU1 gene in Saccharomyces cerevisiae (Blanco, 1994). The PGU1 gene is absent in some S. cerevisiae strains and consequently these strains do not possess polygalacturonase activity (Fernández-González, 2004). Some of the strains that possess a functional PGU1 gene lack activity because the gene is not expressed (Jia & Wheals, 2000). The frequency at which a gene is transcribed is determined by its promoter sequence and by the presence of transcription factors that bind to this promoter in order to activate transcription. Transcription factors that activate the expression of the PGU1 gene are regulated by the mating and filamentous growth mitogen activated protein kinase (MAPK) pathway (Madhani, 1999). Gene expression is, however, not only affected by the promoter sequence of a gene but also by its position in the genome (Flagfeldt, 2009).

PGU1 is located at position 722806..723891, within 25 kb of the right telomere of chromosome X and is therefore subtelomeric (Louw, 2009).

Transcription of genes inserted near telomeres is frequently repressed by an epigenetic effect known as the telomere position effect (TPE) (Vega-Palas, 2000). Packaging of genes into a condensed heterochromatin-like structure at the telomeric tract and subtelomeric region can result in silencing of genes irrespective of their promoters (Martin, 2004). In S. cerevisiae, the telomeres consist of tandem (C1–3A) repeats approximately 350 bp in length. Together with the telomere silencing factors, the telomeres form a non-nucleosomal DNA–protein complex called the telosome (Wright & Shay, 1992). Rap1p, the sequence-specific duplex DNA-binding protein, is the major protein in the telosome. Rap1p binds to the DNA repeat sequences and interacts with other proteins including Rif1p, Rif2p, Sir2p, Sir3p and Sir4p. These proteins interact with each other and subtelomeric nucleosomes via N-termini of histones H3 and H4, thus packaging the telomeric region into a complex heterochromatin-like structure (Vega-Palas, 2000). Genes are silenced by the silence information regulator proteins (Sirp) binding up to 8 kb from the telomeres (Wyrick, 1999). Unlike the telomeric tract, subtelomeric DNA is organized into nucleosomes (Tham & Zakian, 2002). Histones can contribute to Sirp-independent silencing of subtelomeric genes upstream of the Sirp-binding region (Wyrick, 1999). According to the histone code hypothesis, combinations of covalent histone modifications lead to varied transcriptional outputs (Dion, 2005). DNA-regulatory sequences thus only provide a partial explanation of gene regulation. Deciphering the complexity of histone modifications is thus essential in understanding transcriptional regulation in the subtelomeric region. A model that explains how post-translational modification of histones regulates the transcription of different chromosomal regions has been proposed (van Leeuwen & Gottschling, 2002; Martin, 2004). The model suggests that a repressor protein can only bind to the N-terminal tail of histones with hypoacylated or hypomethylated lysine residues, because the presence of these functional groups would inhibit binding of the protein. The repressor protein would thus be restricted to regions of the chromosome in which histones are hypomethylated and hypoacetylated such as subtelomeric regions (Martin, 2004). Gene silencing by TPE in yeast has mainly been demonstrated by inserting reporter genes in close proximity of a telomere (Gottschling, 1990). The only naturally occurring yeast genes that TPE has been demonstrated for are: (1) HMR, the silenced copy of the mating type locus on chromosome III (Thompson, 1994); (2) the TY5-1 retrotransposon, (3) YFR057W, an uncharacterized ORF located adjacent to the telomere on R-VI (Vega-Palas, 2000); and (4) IMD1, a pseudogene encoding an IMP dehydrogenase ortholog located next to the right telomere of chromosome I (Barton & Kaback, 2006). Different methods were used to demonstrate that these genes are silenced by TPE. HMR was found to be derepressed when the H3 histone was mutated and the gene was moved away from the telomere. The H3 mutation did not lead to a significant increase in the transcription of HMR when the gene was integrated adjacent to a telomere (Thompson, 1994). TY5-1 and YFR057W expressions were derepressed by mutating genes that alleviate TPE (i.e. SIR2, SIR3 and SIR4) (Vega-Palas, 2000). IMD1 expression was derepressed by removing the telomere adjacent to this gene from chromosome I; the expression of this gene also increased in a sir3Δ mutant (Barton & Kaback, 2006).

Both pectin degradation and filamentous growth phenotypes have been implicated in S. cerevisiae functioning as a plant pathogen (Juana, 2001; Gognies & Belarbi, 2002). These two phenotypes are coregulated by the same regulatory pathway (Madhani, 1999). It has been shown that the FLO genes, responsible for filamentous growth, are under genetic and epigenetic control (Halme, 2004). Four of the five FLO genes regulating cell surface variation are located in subtelomeric positions 10–40 kb from telomeres and have been shown to be under epigenetic control (Halme, 2004). PGU1 is also subtelomeric. Deletion of five genes DLS1, ISW2, SIR3, HTS3 and ESC1 involved in gene silencing by TPE in S. cerevisiae results in an increase in polygalacturonase activity, indicating that PGU1 transcription might be inhibited by telomeric silencing in the S. cerevisiae BY4742 strain (Louw, 2009).

In this study, we investigate whether PGU1 transcription is also under epigenetic control by shifting the PGU1 gene with its native regulatory sequences to a different position in the genome, away from its telomere, and comparing PGU1 transcription and polygalacturonase activity with that of the wild-type strain. To determine whether the chromatin remodeling complex DLS1 only inhibits polygalacturonase activity in the BY4742 strain, the gene is knocked out in other strains normally devoid of polygalacturonase activity and the strains are screened for recovery in polygalacturonase activity. In order to identify histone modifications that affect PGU1 transcription, microarray data of studies in which different histones were mutated are analyzed.

Materials and methods

Strains, plasmids and culture conditions

The bacteria and yeast strains used in this study are summarized in Table 1.

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Microbial strains used in this study

StrainsGenotype/descriptionReferences
S. cerevisiae strains
BY4742 (S288C background)MATα; his3-1; leu2Δ0; lys2Δ0; ura3Δ0Brachmann (1998)
BY4742 hst3Δ0MATα; his3-1; leu2Δ0; lys2Δ0; ura3Δ0; hst3kanMX4Brachmann (1998)
BY4742 isw2Δ0MATα; his3-1; leu2Δ0; lys2Δ0; ura3Δ0; isw2kanMX4Brachmann (1998)
BY4742 sir3Δ0MATα; his3-1; leu2Δ0; lys2Δ0; ura3Δ0; sir3kanMX4Brachmann (1998)
BY4742 dls1KanMX4MATα; his3-1; leu2Δ0; lys2Δ0; ura3Δ0; yjl064w∷kanMX4Brachmann (1998)
BY4742 pgu1Δ0MATα; his3-1; leu2Δ0; lys2Δ0; ura3Δ0; pgu1kanMX4Brachmann (1998)
FY23MATaura3-52 trp1-63 leu2-1 GAL2Winston (1995)
FY23 dls1KanMX4MATa ura3-52 trp1-63 leu2-1 GAL2; yjl064w∷kanMX4This study
FY23 pgu1Δ0MATaura3-52 trp1-63 leu2-1 GAL2 pgu1kanMX4This study
FY23-1MATaura3-52 trp1-63 leu2-1 GAL2 pgu1kanMX4; URA3PGU1This study
W303-1AMATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15Veal (2003)
W303-1A dls1KanMX4MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15, yjl064w∷kanMX4This study
∑1278bMATα; ura3-52; trp1hisG; leu2hisG; his3hisGvan Dyk (2005)
∑1278b pgu1Δ0MATα; ura3-52; trp1hisG; leu2hisG; his3hisG; pgu1kanMX4This study
∑1278b-1MATα; ura3-52; trp1hisG; leu2hisG; his3hisG; pgu1kanMX4, URA3PGU1This study
Escherichia coli strain
DH5α[F-ϕ80lacZΔM15Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17(rk−, mk+) phoA supE44 thi-1 gyrA96 relA1λ]GIBCO-Invitrogen Life Technologies, Mowbray, South Africa

Plasmids were constructed and amplified in Escherichia coli DH5α grown in Luria–Bertani medium (Biolab diagnostics, Wadenville, South Africa). The medium was supplemented with 100 mg L−1 ampicillin for the selection of resistant bacteria when appropriate.

Saccharomyces cerevisiae wild-type strains were grown in yeast peptone dextrose broth (Biolab diagnostics) at 30 °C on a rotary shaker at 150 r.p.m. Yeast transformants were grown in synthetic complete (SC) medium containing 6.7 g L−1 yeast nitrogen base (YNB) (Difco Scientific Group, Waterfall Park, South Africa) and 20 g L−1 glucose supplemented with the appropriate amino acids to apply auxotrophic pressure. Geneticin-resistant transformants were selected on appropriate media supplemented with 200 mg L−1 geneticin (Sigma-Aldrich, St. Louis). Solid media contained 20 g L−1 agar. Bacteria and yeasts were cultured at 37 and 30 °C, respectively.

Yeast strain construction

The wild-type yeast strains ∑1278b, W303-1A and FY23 (S288C genetic background) were used for the construction of recombinant strains. Deletion mutants were generated by transforming the wild-type strains with PCR-generated knockout cassettes (Table 2). Each gene disruption was replaced with a KanMX4 module (Wach, 1994). Deleting YJL064W resulted in a partial deletion of DLS1, generating the dls1KanMX4 genotype. To move the PGU1 gene to another locus, the pgu1Δ strains were transformed with the YIplac211-PGU1 plasmid linearized with the restriction enzyme ApaI (Roche Diagnostics, Randburg, South Africa), generating FY23 pgu1Δ0, ura3PGU1 (FY23-1) and ∑1278b pgu1Δ0, ura3PGU1 (∑1278b-1). All strains were transformed by electroporation (Volschenk, 2004). Deletion of genes with the KanMX4 knockout cassettes was verified by PCR analysis (results not shown). Integration of PGU1 into the URA3 locus was verified by Southern blot analysis. Genomic DNA was isolated from the S. cerevisiae strains analyzed (Ausubel, 1995). DNA was digested with PstI and ClaI (Roche Diagnostics) separated in a 1% w/v agarose gel and blotted to a nylon membrane, positively charged (Roche Diagnostics). λ DNA digested with the BstEII restriction enzyme (Roche Diagnostics) was loaded as a molecular weight marker. Southern hybridizations were carried out as described in the digoxygenin (DIG) Application Manual (Roche Molecular Biochemicals). A DIG-labeled probe was prepared for the detection of PGU1 by PCR labeling using the PCR DIG probe synthesis kit (Roche Diagnostics). λ DNA was labeled using the DIG DNA labeling kit (Roche Applied Science) to probe the molecular weight marker.

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List of primers used for PCR amplification in this study

Primer nameSequence 5′–3′TemplatePCR purpose and locus amplifiedReferences
5′YJL064w knockoutTCAGTAACGTCTTCGTCGTCGTCTTCGTBY4742 Δyjl064wConstructing knockout cassette Δyjl064w∷kanMX4This study
3′YJL064w knockoutTTGAAAGAACAGCGCTAACAATGTGCBY4742 Δyjl064wConstructing knockout cassette Δyjl064w∷kanMX4This study
PGL1PROMAAGCTTGGACAAGTCGACTTGTCCTGCBY4742 Δpgu1Constructing knockout cassette Δpgu1kanMX4Divol & van Rensburg (2007)
PGU-nat-term-revCGAACTATGGCGAAGGTTGATGAGABY4742 Δpgu1Constructing knockout cassette Δpgu1kanMX4This study
5′PGU1+1600 bp FLANKSGGGTTCCCTGAAGAAACAGAGAATBY4742Amplifying native PGU1 expression cassetteThis study
3′PGU1+1600 bp FLANKSCAATCTTGCTCTTTTCCAACGBY4742Amplifying native PGU1 expression cassetteThis study
5′PPGL1-BGLII pP2AGATCTATGATTTCTGCTAATTCATTACTTATTTCCBY4742Preparing DIG labelled dUTP probe for PGU1Louw (2009)
3′PPGL1-BGLII pP2CTCGAGTTAACAGCTTGCACCAGATCCAGATGBY4742Preparing DIG labelled dUTP probe for PGU1Louw (2009)
Act fwTACCGGCCAAATCGATTCTCBY4742,Σ1278b, FY23, FY23-1, Σ1278b-1qRT ACT1Divol (2006)
Act revCACTGGTATTGTTTTGGATTBY4742, Σ1278b, FY23, FY23-1, Σ1278b-1qRT ACT1Divol (2006)
Pgu1fw2GTGCTTCGGGACATACCATTBY4742, Σ1278b, FY23, FY23-1, Σ1278b-1qRT PGU1Divol & van Rensburg (2007)
Pgu1rev2CGTCAACGCCAACTTTACAABY4742, Σ1278b, FY23, FY23-1, Σ1278b-1qRT PGU1Divol & van Rensburg (2007)

Constructing knockout and integration cassettes

The knockout cassettes for YJL064W and PGU1 were obtained through PCR amplification of the corresponding disrupted genes of the mutants from the BY4742 (Brachmann, 1998) mutant collection supplied by European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF, http://web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).

The PGU1 expression cassette was amplified using PCR. Primers were synthesized by Integrated DNA Technologies (Coralville, IA). All genes were amplified from genomic DNA, using an Applied Biosystems 2720 thermal cycler. Takara ExTaq enzyme and Takara buffer with MgCl2 were used (Separations, Randburg, South Africa). The reaction mixture contained 250 μM of each nucleotide (dNTP), 200 ng DNA, 0.25 μM of each primer and 0.2 mM MgCl2. The primers used are listed in Table 2.

The PGU1 expression cassette was digested with XhoI and HindIII (Roche Diagnostics) and ligated into the corresponding sites of the Yeast Integrating Plasmid, YIplac-211 (Gietz & Sugino, 1988), to generate the plasmid YIplac211-PGU1. Correct cloning was verified by restriction analysis and sequencing (Central Analytical Facility, Stellenbosch University).

Screening for polygalacturonase activity

Strains were screened for polygalacturonase activity using a modified plate assay described by Masoud & Jespersen (2006). All the yeast strains listed in Table 1 were screened for polygalacturonase activity. Polygalacturonase activity could be seen as halos surrounding colonies where polygalacturonic acid was degraded. Five microliters of an overnight culture containing 104 cells was spotted on polygalacturonase plates [1.25% polygalacturonic acid (Sigma-Aldrich), 0.67% YNB, 1% glucose (w/v) (Merck), 2% agar (w/v) (Difco Scientific Group), 0.68% potassium phosphate, pH 4.0] and incubated for 3 days at 30 °C. Degradation halos were visualized by washing colonies off with distilled water and staining plates with 6 M HCl.

Quantitative real-time PCR (qRT-PCR)

RNA isolation

Total RNA was isolated from S. cerevisiae strains FY23 and FY23-1 as described in the hot phenol RNA isolation protocol (Ausubel, 1995). For each strain, a single colony was inoculated into 5 mL SC media and incubated in a roller drum overnight at 30 °C. The cells were pelleted by centrifugation at 5000 g for 5 min. The pellet was washed in water and used to inoculate 10 mL of SC. Cells were harvested during the logarithmic growth phase at an OD600 nm of 1.0.

The quality and concentration of RNA were evaluated by gel electrophoresis and by measuring absorbance; A260 nm was determined for quantification and the 260/280 nm ratio to determine purity.

cDNA synthesis

RNA samples were treated with DNAse I (Roche Diagnostics) to remove any residual DNA contamination, following the manufacturer's instructions. The absence of DNA was confirmed by end-point PCR. One microgram of each RNA sample was converted into cDNA using the ImProm-II Reverse Transcription System (Promega, Madison) following the manufacturer's instructions. Total RNA was used as a template.

qRT-PCR

qRT-PCR was performed on cDNA samples originating from two independent replicate experiments. The experiments were carried out using SYBR-Green dye in a 7500 Real-Time PCR System (Applied Biosystems, Johannesburg, South Africa). Reactions contained Power KAPATaq Ready Mix (KAPA Biosystems, Cape Town, South Africa), forward and reverse primers (0.1 μM each; Table 2) and 20 ng cDNA template. The primers described in Table 2 were used. For each PCR product, melting curves were determined according to the ABI guidelines, ensuring specific amplification of the target gene. Quantitative values were obtained as the threshold PCR cycle number (Ct) when the increase in the fluorescent signal of the PCR product showed exponential amplification. Transcription of each gene was normalized to that of ACT1 in the same sample. The cycle threshold (Ct) value for each reaction was determined using the Sequence Detection System, 7500 real-time pcr system software package (Applied Biosystems). Ct values were used to calculate the expression of PGU1 in the recombinant strains relative to that of the same gene in the wild-type strain. Fold change was calculated via the Embedded Image method for each sample in triplicate, in which 1 indicates no change in abundance (Livak & Schmittgen, 2001).

Results

Polygalacturonase activity in S. cerevisiae deletion mutants

Derepression of polygalacturonase activity in the following BY4742 mutants listed in Table 1: dls1KanMX4, isw2Δ, sir3Δ, hst3Δ and esc1Δ was compared by plate assays. Disruption of DLS1 and ISW2 resulted in the most significant increase in polygalacturonase activity, the hst3Δ mutant had medium polygalacturonase activity and the sir3Δ and esc1Δ mutants showed a slight recovery in polygalacturonase activity (results not shown). To determine whether DLS1 acts as repressor of polygalacturonase activity in other strains lacking polygalacturonase activity, W303-1A dls1KanMX4 and FY23 dls1KanMX4 mutants were generated. Polygalacturonase activities of these two mutant strains and the BY4742 Δdls1KanMX4 strain obtained from the EUROSCARF deletion mutant library were compared with the corresponding wild-type strains (Fig. 1).

1

Polygalacturonase plate assay comparing polygalacturonase activity between (a) BY4742 dls1KanMX4, (b) BY4742, (c) FY23 dls1KanMX4, (d) FY23, (e) W303-1A dls1KanMX4 and (f) W303-1A. Deletion of the DLS1 gene resulted in the recovery of polygalacturonase activity in all three strains.

It can be seen from Fig. 1 that the BY4742, FY23 and W303-1A wild-type strains showed no polygalacturonase activity. Deletion of the DLS1 gene, however, conferred polygalacturonase activity upon all three strains.

Shifting the PGU1 gene to a different locus

To confirm that transcription of the PGU1 gene was inhibited by an epigenetic effect in some S. cerevisiae strains, the gene with its native promoter and terminator was shifted to a different (nontelomeric) locus. PGU1 was deleted in locus YJR153w with a KanMX4-containing cassette in strains FY23 (isogenic to BY4742) and ∑1278b; deletion was confirmed by PCR (results not shown). YIplac211-PGU1 was subsequently integrated into URA3, generating FY23, pgu1kanMX4; URA3PGU1 (FY23-1) and ∑1278b, pgu1kanMX4; URA3PGU1 (∑1278b-1). The presence of a single copy of PGU1, shifted from the YJR153W locus on chromosome X to the YEL021W locus on chromosome V, was confirmed by Southern blot analysis (Fig. 2).

2

Southern blot analysis of genomic DNA isolated from the FY23 and ∑1278b wild-type strains and FY23-1 and ∑1278B-1 recombinant strains, probed for the PGU1 gene, showing a single copy of PGU1 present in all strains, but located in different loci for wild-type and recombinant strains. Lane a, marker lane (λ DNA digested with BstEII); lane b, FY23; lane c, FY23-1; lane d, ∑1278b and lane e, ∑1278b-1.

Polygalacturonase activity in strains in which the PGU1 gene has been shifted to a different locus

The polygalacturonase activity of the wild-type strains was compared with the mutant strains in which PGU1 was shifted to a different locus (Fig. 3). From Fig. 3, it can be seen that FY23, an S. cerevisiae strain without polygalacturonase activity, was able to degrade polygalacturonic acid when PGU1 was shifted to a nontelomeric locus, generating FY23-1. When ∑1278b, an S. cerevisiae strain with polygalacturonase activity, was engineered in the same manner; the resulting recombinant strain ∑1278b-1 showed polygalacturonase activity similar to that of the wild-type strain.

3

Polygalacturonase plate assay comparing polygalacturonase activity between (a) FY23-1, (b) FY23, (c) ∑1278b-1 and (d) ∑1278b. Shifting the PGU1 gene to a different position in the genome, away from its telomere, results in an increase in polygalacturonase activity in the FY23-1 strain, while the ∑1278b-1 maintained its activity.

Transcription of PGU1 in FY23-1

qRT-PCR was carried out to confirm that the recovery in polygalacturonase activity in the FY23-1 strain was due to an increase in transcription when the gene was shifted to a different locus. Transcription of PGU1 increased 2.34-fold (±0.14) in the FY23-1 strain compared with the FY23 wild-type strain.

Discussion

Polygalacturonase activity has been shown to be regulated by transcription of the PGU1 gene. The gene has, however, been shown to have an identical promoter region in strains with and without polygalacturonase activity (Jia & Wheals, 2000). DNA-regulatory sequences provide only a partial explanation of gene regulation (Martin, 2004). Screening the S. cerevisiae genome for activators and inhibitors of polygalacturonase activity revealed that five genes implicated in telomeric silencing (i.e. DLS1, ISW2, SIR3, HST3 and ESC1) are involved in regulating PGU1 transcription (Louw, 2009). Because DLS1 deletion resulted in the most significant recovery in polygalacturonase activity in BY4742 among the mutants identified by the deletion library screen (results not shown), we focused on this chromatin remodeler as an inhibitor of polygalacturonase activity in other strains that lack polygalacturonase activity. In this study, we proved that PGU1 transcription is indeed influenced by an epigenetic effect. Moving the PGU1 cassette from its subtelomeric position at locus YJR153W on chromosome X to YEL021W (URA3) on chromosome V enabled FY23, an S. cerevisiae strain without polygalacturonase activity, to degrade polygalacturonic acid. RT-PCR analysis confirmed that this recovery in activity was due to a doubling in PGU1 transcription. This result was in agreement with that of Flagfeldt (2009), who found an increase in transcription when shifting a marker gene from a subtelomeric position to the URA3 locus.

The position of PGU1 in the genome could possibly explain epigenetic silencing in some strains. Post-translational modification of histones leads to the division of chromosomes into silenced and transcribed regions (Shahbazian & Grunstein, 2007). PGU1 is located at a subtelomeric position ∼25 kb from the right telomere of chromosome X. It has been shown by Wyrick (1999) that genes located in this region can be silenced due to post-translational modification of histones.

PGU1 is located beyond the 3–4 kb extent from the telomere within which genes can be silenced by Sirp binding. A slight increase in polygalacturonase activity was, however, found upon deletion of SIR3 by Louw (2009) and was confirmed in this study.

The SIR3 deletion can result in an increase in polygalacturonase activity by an indirect effect. Microarray analysis showed that a deletion of SIR3 results in a 65.9-fold decrease in FUS3 transcription (Wyrick, 1999). FUS3 is the MAPK in the mating MAPK pathway and has been shown to act as an inhibitor of PGU1 (Madhani, 1999). FUS3 is not involved in epigenetic regulation, but the decrease in FUS3 expression could potentially lead to an increase in PGU1 transcription. Fus3p induces ubiquitination of Tec1p, the main transcription factor regulating PGU1 transcription, through the SCFCdc4 ubiquitin ligase during mating (Chou, 2004). A decrease in Fus3p will thus result in more Tec1p being available in the cell, resulting in a higher expression of PGU1.

To determine whether the chromatin remodeling complex DLS1 inhibits polygalacturonase activity in all strains, regardless of their ability to express PGU1, DLS1 was also mutated in two other strains: FY23 and W303-1A. These two strains possess PGU1, but display no polygalacturonase activity. From Fig. 1, it can be seen that BY4742 dls1KanMX4, FY23 dls1KanMX4 and W303-1 dls1KanMX4 recovered polygalacturonase activity, while the corresponding wild-type strains could not degrade polygalacturonic acid. Dls1p plays a critical role in Isw2p-dependent repression of a wide variety of genes in vivo (McConnell, 2004). DLS1 has been shown to be required for Isw2p-dependent chromatin remodeling of genes at the telomeric regions (Iida & Araki, 2004). Chromatin remodelers can restructure, mobilize or eject nucleosomes, allowing exposure of DNA in chromatin (Cairns, 2007). ISW2 is an ortholog of the human chromatin accessibility complex and functions by sliding mononucleosomes from the end to the center of DNA (Dang, 2007). Chromatin analysis revealed that Dls1p is required in the formation of repressive chromatin structure subsequent to cross-linking of Isw2p with chromatin (McConnell, 2004). In this study, it has been shown that PGU1 transcription is inhibited due to its position in the genome of strains lacking polygalacturonase activity. Because DLS1 and ISW2 are required for the formation of a repressive chromatin structure and deletion of either one of these genes results in derepression of polygalacturonase activity, it indicates that PGU1 transcription is inhibited due to ISW2p-dependent formation of a repressive chromatin structure at the native PGU1 locus YJR153w in strains possessing the PGU1 gene, but lacking polygalacturonase activity. Deletion of the ISW2 gene has been shown to result in Flo11p-independent invasive growth (Trachtulcova, 2004). The phenotypes invasive growth and polygalacturonase degradation are coregulated by the invasive growth KSS1 MAPK pathway, activating the transcription factors Tec1p and Ste12p. It seems that a mutation affecting both phenotypes would do so by affecting this regulatory pathway. This is, however, not the case because invasive growth increases in Δkss1Δisw2 and Δtec1Δisw2 mutants (Roberts, 2000). It has been shown that the Isw2–Itc1 complex could play a role in reorganizing genomic expression during metabolic adaptation to starvation conditions (Kent, 2001). Pectolytic activity and invasive growth are coordinated foraging behaviors under starvation conditions by the yeast (Madhani, 1999). The regulation of transcription of the genes responsible for these phenotypes by the same chromatin remodeling complex shows that these genes are not only regulated by the same transcription factors, but are also regulated on an epigenetic level.

TPE is the transcriptional repression of genes located near telomeres (Mondoux & Zakian, 2007). Telomeres facilitate silencing of nearby promoters through propagation of the Sir silencing complex (Perrod & Gasser, 2003). Histones have, however, been shown to make Sir-independent contributions to telomeric silencing (Wyrick, 1999). Histone N-terminal domains play important roles in regulating chromatin structure and gene transcription (Parra, 2006). Genomic chromatin structure is organized into condensed, silent heterochromatin and more relaxed active euchromatin regions. The nucleosome is the repeating unit in chromatin and consists of two copies of each of the four histone proteins, forming the histone octamer, with 147 bp of DNA wrapped in nearly two turns of a superhelix around the core octamer (Shahbazian & Grunstein, 2007).

Post-translational modification of histones has been proposed to demarcate regions of heterochromatin and euchromatin in S. cerevisiae (Martin, 2004). Previously published microarray data sets were investigated in order to determine the influence of different histones on the chromatin structure that influence PGU1 transcription (Wyrick, 1999; Martin, 2004; Parra, 2006; Parra & Wyrick, 2007).

Table 3 shows the effect that the mutation of different histones had on PGU1 transcription. From this table, it can be seen that the N-terminal domains of H3 and H4 play a role in the repression of PGU1 expression. Mutating different residues in the N-terminal tails of H2A and H2B had no effect on PGU1 transcription. Changing lysines 9, 14, 18 and 23 to glysine in the N-terminal tails of H3 resulted in a 2.13-fold increase in PGU1 transcription compared with the wild-type strain. Changing lysines 5, 8, 12 and 16 to glysine in the N-terminal tails of H4 resulted in a 5.18-fold increase in PGU1 transcription compared with the wild-type strain, and depletion of this histone resulted in the largest increase in PGU1 transcription (Table 3). PGU1 expression has been shown to increase 20-fold upon depletion of the histone H4. Because PGU1 expression seems to be silenced due to the proximity of the gene to the right telomere of chromosome X, it would be expected that genes located between PGU1 and the telomere would also be silenced. Table 4 shows the increase in the transcription of genes located in the subtelomeric region of X–R, upon depletion of H4. It can be seen from Table 4 that transcription of the cluster of genes located between PGU1 and the telomere was derepressed upon depletion of H4 (Wyrick, 1999). Analyzing genomewide gene expression, Martin (2004) found transcriptional repression to be dependent on the histone H3 N-terminal domain, but not the histone H4 N-terminal domain. In contrast to this analysis of the complete genome, analyzing microarray results by focusing on the X–R subtelomeric region, we found transcriptional repression of PGU1 to be dependent on the H4 N-terminal domain and transcription of all subtelomeric genes on chromosome X to increase upon depletion of H4.

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Fold change in PGU1 transcription upon mutation of different histones

MutationResulting fold change increase in PGU1 transcriptionReferences
H2A N-terminal tailNoneParra & Wyrick (2007), http://wyrick.sbs.wsu.edu/histoneH2A/
H2B N-terminal tailNoneParra (2006), http://wyrick.sbs.wsu.edu/histoneH2B/
H3 N-terminal tail (lysine-9, -14, -18, -23 to glysine)2.13Martin (2004), http://wyrick.sbs.wsu.edu/histoneH3/
H4 N-terminal tail (lysine-5, -8, -12, -16 to glysine)5.18Martin (2004), http://wyrick.sbs.wsu.edu/histoneH3/
H4 depletion19.38Wyrick (1999), http://jura.wi.mit.edu/cgi-bin/young_public/lists.cgi
View this table:
4

Increase in the transcription of genes located between PGU1 and the telomere X–R upon depletion of the histone H4 according to Wyrick (1999)

GeneDistance located from the right telomere of chromosome X (kb)Increase in transcription (fold change)
YJR162C0.499.1
COS51.751.41
YJR160C5.932.7
SOR19.73.7
HXT161313.3
YJR157W15.2314.3
THI1116.155.8
AAD1018.343.2
YJR154W19.961.5
PGU122.8819.38

Our results showed that PGU1 was silenced due to its subtelomeric position on chromosome X. The Isw2p chromatin remodeling complex and its histone fold motif containing subunit Dls1p are important in establishing this repressed state. Transcriptional repression of PGU1 is dependent on the H4 N-terminal domain as microarray analysis indicates that H4 is involved in inhibition of transcription of all the genes in the subtelomeric area of chromosome X.

Footnotes

  • Editor: Isak Pretorius

References

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