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Mol Cell Biol. Mar 2002; 22(5): 1298–1306.
PMCID: PMC134702

Set2 Is a Nucleosomal Histone H3-Selective Methyltransferase That Mediates Transcriptional Repression

Abstract

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.

Eukaryotic DNA is complexed in cells by histone proteins to form the fundamental repeating unit of chromatin, the nucleosome. Stretches of nucleosomes are further folded upon themselves to create higher-order chromatin structures that are currently not well defined. Compaction of DNA in this manner imposes a severe impediment to proteins that require access to the DNA template. Clear examples of this impediment have been shown to exist for the machinery that drives DNA transcription (28, 38, 41). However, this same impediment faces all aspects of DNA metabolism, including replication, repair and recombination (18, 40).

Posttranslational modifications of histone amino termini are recognized to play a central role in the control of chromatin structure and function. A diverse array of covalent histone modifications have been documented that take place on the tail domains of histones which protrude away from the nucleosome (9, 39). We and others have proposed that these modifications form a histone code which directly regulates chromatin function either by altering the specific structure of the chromatin polymer itself and/or by recruiting proteins or protein complexes that uniquely recognize a single or combinatorial set of modifications on one or more histone tails (14, 35, 37). For example, recent evidence showing that the bromodomains of various histone acetyltransferases, including PCAF, GCN5 and TAFII250, bind to acetylated lysines in the histone tails suggests that specific recruitment of the transcriptional apparatus to promoters is one likely mechanism to explain how histone modifications influence transcription (8, 22). It appears that other histone modifications, including methylation, function in the same manner (see below).

Histone methylation is a posttranslational modification that occurs on lysine and arginine residues in the H3 and H4 tail domains (reviewed in reference 42). In histone H3, lysines 4, 9, 27, and 36 are well-documented sites of methylation, while in histone H4, lysine methylation is restricted to lysine 20 (36, 39). Recent work has identified the Su(var)3-9 family as being H3 lysine 9-specific histone methyltransferases (HMTs). These proteins, which play an important role in the establishment and maintenance of heterochromatin, mediate their methylation activity through the SET domain. Given that this methylation mark is specifically recognized by the chromo domains of HP1 and its Schizosaccharomyces pombe homologue Swi6 (2, 7, 12, 19, 25), it is proposed that specific recruitment of these proteins in a lysine 9 (Lys9)-dependent manner mediates the establishment of heterochromatin (13, 14, 32). Outside of heterochromatin-mediated silencing, transcriptional repression by retinoblastoma has recently been shown to require both SUV39H1 and HP1 (26), thus providing a novel mechanism by which the general repression of euchromatic genes may be established (14, 32).

In this study, we sought to identify and characterize histone-specific methyltransferases from Saccharomyces cerevisiae. Using a biochemical approach, we identified two separate nucleosome-specific HMT activities, one of which contained the SET family member Set2. The HMT activity of Set2, mediated by its SET domain, is specific for nucleosomal substrates and is site-selective for lysine 36 (Lys36) in histone H3. Generation of an H3 Lys36 antiserum reveals that Set2 is the only SET domain-containing protein in S. cerevisiae responsible for mediating methylation at this site. Finally, tethering experiments show that in vivo, Set2 functions as a transcriptional repressor that mediates its repression partially through the SET domain. These results suggest a role for Set2 and its methylation activity in regulating the repression of gene transcription.

MATERIALS AND METHODS

S. cerevisiae strains.

Purification of Set2 was carried out using the S. cerevisiae strain CY396, which is described elsewhere (29). The SET domain deletion strains and isogenic wild-type (WT) strain (in the BY4742 background) were obtained from Research Genetics. The set1Δ and isogenic wild-type strains are described elsewhere (3, 3a). For in vivo repression assays, the wild-type S. cerevisiae strain YMH171, a strain previously employed for repression assays (43), was used.

Protein purification and chromatography.

Whole-cell extracts were prepared from 20 liters of the S. cerevisiae strain CY396 grown to mid-log phase as described (10). This extract (160 ml total) was bound batchwise with 20 ml of Ni2+-nitrilotriacetic acid (NTA)-agarose (Qiagen), and the resin was washed in a column with 20 mM imidazole-containing extraction buffer (10). Proteins were eluted with 300 mM imidazole and passed through a Mono Q HR 5/5 column (Amersham Pharmacia Biotech). The flowthrough was then collected and loaded directly onto a Mono S HR 5/5 column (Amersham Pharmacia Biotech) before bound proteins were eluted using a 25-ml linear gradient from 0.1 to 1 M NaCl. Three microliters from every other fraction was assayed for HMT activity as described below.

Peak 1 and peak 2 HMT activities were pooled, concentrated to 0.5 ml using a Centriprep-30 (Amicon), and then loaded on a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated with extraction buffer plus 0.5 mM dithiothreitol (DTT). The chromatographic system was calibrated using the following standard proteins: thyroglobulin (669 kDa), apoferritin (440 kDa), aldolase (158 kDa), and ovalbumin (45 kDa). Peak 1 and peak 2, which comigrated between 100 and 200 kDa, were pooled and diluted to 0.1 M NaCl prior to loading on a 0.5-ml heparin-Sepharose column (Sigma). Bound proteins were eluted with a 5 ml of a 0.1 to 1 M NaCl gradient. Peak HMT activity was pooled and then loaded on the Mono S HR 5/5 column as described above. Peak 1 and 2 activities were pooled and loaded on a Mini S PC 3.2/3 column (Amersham Pharmacia Biotech) attached to a SMART System (Amersham Pharmacia Biotech).

To further purify peak 1 for mass spectrometry identification, peak 1 activity from the Mini S column was pooled, concentrated to ≤50 μl as described above, and then loaded onto a Superose 12 PC 3.2/30 column (Amersham Pharmacia Biotech) equilibrated to 300 mM NaCl. Then 5 μl of each fraction (50 μl) was analyzed for HMT activity as described below. The Superose 12 column was calibrated using the following standard proteins: thyroglobulin (669 kDa), apoferritin (440 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). We note that attempts to resolve bacterially produced Set2-Flag (see below) on the Superose 12 column to determine the homo- or heterodimeric nature of this protein have been unsuccessful due to significant levels of aggregation observed under the isolation conditions used to purify Set2 (data not shown).

Cloning and expression of Set2.

The full-length Set2 gene was amplified from S. cerevisiae genomic DNA by PCR using an oligonucleotide primer that added a BamHI site in front of the ATG start site (5"-CGCGGATCCATGTCGAAGAACCAAAGTGTGAG-3") and an oligonucleotide primer that added a SalI site that followed a Flag coding sequence and stop site (5"-ACGCGTCACTTACTTGTCATCGTCGTCCTTGTAGTCTGATGATGTTGAAGGTGGAGGAG-3"). Set2-Flag PCR product was spin column purified using the QIAquick PCR purification kit (Qiagen), digested with BamHI and SalI, agarose gel purified, and then cloned into the Escherichia coli expression vector pCAL-n (Stratagene). The Set2 coding region was sequenced for accuracy. The resulting expression plasmid was transformed and expressed in the BL21-Gold(DE3) E. coli strain (Stratagene).

Cells containing vector only or Set2-Flag (500 ml each) were grown to an optical density at 600 nm (OD600) of 0.8, followed by addition of 0.1 mM isopropyl-d-thiogalactoside for 3 h at 30°C. Harvested cells were resuspended in 30 ml of lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT, 1.0 mM magnesium acetate, 2 mM CaCl2, 0.1% NP-40, 5 mg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 2 μg of leupeptin, aprotinin, and pepstatin per ml each) and sonicated. Cell debris was centrifuged at 14,000 × g, and the supernatant was incubated batchwise with 200 μl of the calmodulin affinity resin (Stratagene). Bound proteins were eluted in a column according to the manufacturer's instructions except that the elution was done with stepwise increases in NaCl (150 mM, 300 mM, and 500 mM; 1 ml at each elution). Protein concentration was determined by Coomassie staining of sodium dodecyl sulfate (SDS)-polyacrylamide gels using bovine serum albumin (BSA) as the standard.

Mutations in the Set domain of Set2 (at R195 and C201) were made by standard PCR mutagenesis methods using the Set2-Flag plasmid. After sequencing through the N terminus and SET domain for accuracy, a BamHI-NsiI fragment encompassing the N terminus and SET domain of Set2 was recloned into the Set2-Flag construct, generating the final mutant expression plasmids. Expression and purification of these proteins were performed as described above. For S. cerevisiae expression, the full-length Set2-Flag and Set2 SET domain mutant constructs described for bacterial expression were digested with BamHI and SalI, and the purified full-length DNAs were subcloned into the p416 alcohol dehydrogenase (ADH) expression plasmid that is driven by the ADH1 promoter (24).

Methyltransferase activity assays.

For HMT assays, partially purified S. cerevisiae extracts or 0.2 μg of recombinantly expressed Set2 or SET domain mutants of Set2 were incubated with either 2 μg of chicken core histones or mononucleosomes (23) along with 0.55 μCi of S-adenosyl-l-[methyl-3H]methionine (3H-AdoMet; 72 Ci/mmol; NEN Life Science Products) in methyltransferase buffer (final concentrations being 50 mM Tris [pH 9.0], 1 mM PMSF, and 0.5 mM DTT) for 30 min at 30°C in a total volume of 10 μl. Then 2 μl of the reaction was spotted on Whatman P-81 for liquid scintillation counting to monitor reactions, while the remainder was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie staining and fluorography.

Set2 site mapping and protein microsequencing.

For labeling studies involving recombinant Set2 for microsequencing, the above HMT reaction volume, using chicken nucleosomes as the substrate, was scaled up 20-fold. Histones from this reaction were acid extracted with 0.4 N H2SO4 and precipitated with trichloroacetic acid (TCA), and the H3 was purified by reverse-phase high-pressure liquid chromatography (RP-HPLC) using a C8 column. 3H-methylated H3 (≈10 μg) was digested with 0.05 μg of V8 protease (Sigma) in 50 mM NH4HCO3 in a 10-μl volume for 3 h at 37°C; digestion products were then separated by RP-HPLC. The H3 1-50 fragment, identified by mass spectrometry as being the only V8 product strongly methylated by Set2, was further digested with 0.5 U of Arg-C protease (Roche Molecular Biochemicals) in a 10-μl reaction volume for 3 h at 37°C; reaction products were again separated by RP-HPLC. The 3H-methylated H3 Arg-C fragment was sequenced in an Applied Biosystems model 477A protein sequencer with an in-line 120A phenylthiohydantoin (PTH) analyzer (Applied Biosystems) using optimized cycles. After conversion, 50% of the sample was transferred to the RP-HPLC for PTH-amino acid identification, and the other 50% was collected for determination of radioactivity by scintillation counting.

Electrophoresis and Western blotting.

SDS-PAGE and Western blot analyses were performed using procedures and reagents from Amersham Pharmacia Biotech. Antiserum detection was made using the ECL+plus chemiluminescent kit (Amersham Pharmacia Biotech). Rabbit anti-LexA (Upstate Biotechnology Inc.) was used at a dilution of 1:3,000, and mouse monoclonal anti-Flag antiserum (M2; Sigma) was used at 1 μg/ml. The anti-H3(Lys36)Me antiserum was routinely used at a dilution of 1:10,000.

Development of an antiserum specific to Lys36 methylation of histone H3.

A synthetic peptide coding for sequence 30 to 38 of the human H3.1 amino terminus (NH2-C*PATGGVKKP), in which Lys36 (underlined) was made dimethyllysine (Bachem), was conjugated by standard protocols to keyhole limpet hemocyanin via an N-terminal artificial cysteine (C*) prior to rabbit immunization. Enzyme-linked immunosorbent assays (ELISAs) were performed as previously described (4) using various amounts of unmodified or Lys36-methylated H3 30-38 peptides (data not shown).

Mass spectrometry.

For the identification of proteins from gel slices, fractions from the Superose 12 size exclusion column containing peak 1 HMT activity were resolved on a SDS-10% PAGE gel followed by silver staining as described (30). Candidate gel bands were excised and digested with trypsin as described (33). Peptides were then extracted from the gel, and an aliquot of the resultant peptides was loaded on a microcapillary column packed with 5-μm C18 particles (YMC ODS-AQ; Waters). The peptides were gradient eluted into an LCQ DECA mass spectrometer (ThermoFinnigan) for data-dependent mass spectral analysis. The HPLC gradient was 0 to 60% B in 70 min, 60 to 100% B in 15 min. Solvents A and B were 0.1 M acetic acid in water and 0.1 M acetic acid in 70% acetonitrile, respectively. Peptide sequences identified from this analysis were used to search the S. cerevisiae database to identify respective candidate proteins.

Transcriptional repression assays.

Full-length Set2 or the Set2 SET domain mutants were amplified by PCR and cloned into a C-terminal LexA DNA-binding domain fusion construct driven by the ADH1 promoter (gift from Mike Hampsey). All constructs were confirmed by sequencing and shown to express full-length products when transformed into S. cerevisiae (see Fig. Fig.6B).6B). Repression assays were performed as previously described (43). In brief, the Set2-LexA constructs, along with an Rpd3-LexA expression construct, were individually transformed into the wild-type S. cerevisiae strain YMH171 along with the lacZ reporter construct (pCK30) that contained one LexA operator site upstream of the CYC1 promoter (17). β-Galactosidase assays were performed as described (1), and the values are reported as the fold repression using the vector-only (LexA DNA-binding domain only) as the comparison. All values represent the average for at least three independent transformants repeated in duplicate. Statistical differences between the level of repression of Set2-LexA, Set2(R195G)-LexA, and Set2(C201A)-LexA were determined using one-way analysis of variance followed by Tukey's post hoc test for individual comparisons (performed using Prism; Graphpad Software Inc.).

FIG. 6.
SET domain point mutants in Set2 are partially defective in transcriptional repression when tethered to a heterologous promoter. (A) The wild-type S. cerevisiae strain YMH171 was transformed with either LexA vector-only, Rpd3-LexA, Set2(WT)-LexA, Set2(R195G)-LexA, ...

RESULTS

Purification of native HMT activities from S. cerevisiae.

A number of studies have documented the existence of both lysine and arginine methylation on histones in vivo (42). However, the functional significance of histone methylation has largely remained obscure, due, in part, to the lack of knowledge regarding the identity of the enzymes that mediate histone methylation. To identify native S. cerevisiae HMTs, whole-cell extracts, prepared from asynchronously growing S. cerevisiae cells, were fractionated over a series of chromatographic steps (Fig. (Fig.1A)1A) that have proven useful in the identification of native histone acetyltransferases (10). Column fractions were then analyzed for histone and nucleosomal HMT activity using chicken core histones or mononucleosomes as the substrate.

FIG. 1.
Partial purification of two nucleosomal H3 HMT activities from S. cerevisiae. (A) Schematic representation of the chromatographic procedure used to partially purify two nucleosome-specific HMT activities from S. cerevisiae whole-cell extracts (see text ...

Preliminary surveys of the Mono S and Mono Q fractions from the unbound nickel-agarose material revealed no significant HMT activities (data not shown). In contrast, analysis of the 300 mM imidazole-eluted material from the nickel-agarose resin revealed the presence of two peaks of HMT activity specific to the Mono S column that were nucleosome and H3 selective (see Fig. Fig.1C1C and and3A3A for examples). No significant HMT activity was observed from nickel-agarose-bound Mono Q fractions (data not shown).

FIG. 3.
Peak 1 HMT activity in S. cerevisiae is dependent on Set2. Whole-cell extracts prepared from S. cerevisiae strains which were either wild type, set2Δ, or set2Δ containing a Set2-Flag expression construct were bound and eluted from nickel-agarose, ...

To further characterize these HMT activities, both peaks of activity from the first Mono S fractionation were further fractionated by Superose 6 and heparin-Sepharose chromatography. Results showed that both peaks were resolved as a single peak of HMT activity on both columns, and size estimation on the Superose 6 column showed these activities to have an apparent native molecular mass of ≈100 to 200 kDa (data not shown). Subsequent purification of these activities through additional cation exchange columns restored the separation of the two individual peaks of nucleosomal H3 HMT activity (Fig. (Fig.1B1B and andC;C; hereafter referred to as peak 1 and peak 2).

Given the greater abundance of HMT activity observed with peak 1 than with peak 2, we attempted to identify the polypeptide responsible for catalytic activity from HMT peak 1. Fractions 12 to 16 (corresponding to peak 1) from the Mini S column were pooled and then subjected to chromatography on a Superose 12 size exclusion column. Results from this separation revealed peak 1 to be approximately 175 kDa (Fig. (Fig.2A),2A), with the majority of HMT activity restricted to fractions 9 and 10 (Fig. (Fig.2B).2B). Comparison of the protein profiles revealed several candidate polypeptides that cofractionated closely with HMT activity. These bands were excised and digested with trypsin, and the resulting peptides were examined by nano-HPLC microelectrospray ionization tandem mass spectrometry. Of the proteins tentatively identified from this analysis, one of these was identified as Set2 (Fig. (Fig.2C2C).

FIG.2.
Set2 is identified as a component of the first peak of nucleosomal HMT activity. (A) Peak 1 HMT activity from the Mini S column was fractionated using a Superose 12 size exclusion column, and collected fractions were analyzed for nucleosome-specific HMT ...

Set2 is required for peak 1 HMT activity.

To further determine if Set2 is necessary for the establishment of peak 1 and/or peak 2 HMT activity, whole-cell extracts were prepared and fractionated as described above except from a yeast strain in which SET2 was disrupted (set2Δ). In parallel, an isogenic WT strain was examined. Both extracts were then analyzed for the presence of the two nucleosome-specific HMT peaks of activity. As shown in the top panels of Fig. Fig.3,3, fractions prepared from the WT strain showed the presence of both peaks of nucleosomal HMT activity, as expected. In contrast, extracts prepared from set2Δ lacked peak 1 but not peak 2 HMT activity, suggesting that Set2 is responsible for peak 1 HMT activity (Fig. (Fig.3,3, middle panels).

To demonstrate the ability of Set 2 to restore peak 1 HMT activity, an expression plasmid in which a Flag epitope was fused to the C terminus of Set2 was constructed and introduced into the set2Δ strain. Results clearly showed that expression of Set2 restores the HMT activity of peak 1 (Fig. (Fig.3,3, lower panels). In agreement, Set2 comigrated precisely in the Mono S fractions that corresponded to peak 1 but not peak 2 HMT activity (Fig. (Fig.3,3, lower panels). These results demonstrate not only that Set2 is required for the HMT activity of peak 1, but also that Set2 is not responsible for peak 2 activity.

Set2 is a nucleosomal HMT highly selective for H3.

To determine if Set2 has intrinsic HMT activity, recombinant Set2 containing an N-terminal calmodulin-binding protein (CBP) and a C-terminal Flag tag was expressed in bacteria. In addition, similar constructs were generated in which two independent amino acid substitutions were generated in the SET domain (R195G and C201A; see Fig. Fig.4A).4A). R195 and C201 are highly conserved residues in the SET domain of Set2, and recently, homologous residues were shown to be important for the HMT activity of SUV39H1 (31). Equivalent amounts of recombinant proteins were purified using calmodulin resin (determined by Coomassie staining and by Western blot analysis, Fig. Fig.4B)4B) and used in standard HMT assays. In agreement with our earlier findings (see Fig. Fig.3),3), WT Set2 protein contained strong nucleosome-specific HMT activity specific for H3 (Fig. (Fig.4C4C and andD).D). In contrast, both SET domain point mutants, R195G and C201A, showed dramatically reduced HMT activity towards nucleosomes (Fig. (Fig.4C).4C). In addition, no significant HMT activity was observed by Set2 on recombinant H3 in either the presence or absence of sheared calf thymus DNA or histone octamers, indicating that a DNA-bound nucleosomal structure is required for Set2 HMT activity (data not shown).

FIG. 4.
Set2 methylation activity is dependent upon the SET domain. (A) Schematic representation of the domain structure of the recombinant Set2 protein expressed in bacteria. Substitutions made of highly conserved amino acids in the SET domain are shown. (B) ...

Set2 is responsible for mediating H3 lysine 36 methylation in vivo.

We next sought to determine the target site(s) of Set2 in H3 both in vitro and in vivo. Unexpectedly, preliminary microsequencing analyses, using H3 from reactions involving chicken nucleosomes and recombinant Set2, revealed no evidence for Set2-mediated methylation in the extreme 1-27 amino terminus of H3 (data not shown). We reasoned that methylation by Set2 might occur elsewhere in the histone tail domain or in the C terminus of H3. To that end, a combination of endoproteinases, Glu-C and Arg-C, were employed to identify H3 peptide fragments that were methylated preferentially by Set2 under our assay conditions. From these stepwise digestions (see Materials and Methods for details), one predominant methylated fragment was isolated and determined by mass spectrometry to be amino acid residues 27 to 40 (data not shown). As shown in Fig. Fig.5A,5A, microsequence analysis of the radiolabled Arg-C H3 peptide showed clearly that Lys36 is the exclusive site of Set2 methylation under these in vitro conditions.

FIG. 5.
Methylation of H3 at lysine 36 is mediated by Set2. (A) RP-HPLC-purified H3 isolated from Set2 reactions involving 3H-AdoMet and chicken nucleosomes was digested with endoproteinase Glu-C, and the resulting peptide fragments were separated by RP-HPLC. ...

To determine if Set2 methylation is responsible for H3 Lys36 methylation in vivo, and to determine if any other SET domain-containing proteins in S. cerevisiae mediate methylation at this site, a novel H3 Lys36 methyl-specific antiserum was generated, α-Me(Lys36)H3, by standard procedures (see Materials and Methods). Using ELISAs, we found the resulting antiserum to be highly selective for the Lys36 methyl-containing H3 peptide, but not for a corresponding unmethylated peptide or H3 peptides methylated at lysine 4 or 9 (data not shown). To further characterize the specificity of the α-Me(Lys36)H3 antiserum, chicken mononucleosomes were incubated with or without recombinant Set2 in the presence of AdoMet, and the products were analyzed by immunoblotting.

As shown in Fig. Fig.5B,5B, nucleosomal H3 from reactions including Set2 was strongly recognized by α-Me(Lys36)H3 compared to control (minus Set2) reactions. Moreover, recognition of nucleosomal H3 by the α-Me(Lys36)H3 antiserum in Set2 reactions was completely abolished by preincubation of the antiserum with the Lys36 methyl-containing peptide (Fig. (Fig.5B).5B). In contrast, preincubation of α-Me(Lys36)H3 with an unmethylated H3 peptide or H3 peptides methylated at lysine 4 or 9 did not prevent the recognition of Set2-dependent Lys36 methylation, demonstrating that this antiserum is selective for H3 Lys36 methylation. In support, the α-Me(Lys36)H3 antiserum did not recognize recombinant Xenopus H3 (Fig. (Fig.5B).5B). Weak recognition of chicken H3 by the α-Me(Lys36)H3 antiserum in control reactions suggests a low level of Lys36 methylation in chickens, a result confirmed in S. cerevisiae, Tetrahymena, and human 293T cells (data not shown).

Next, we sought to identify the enzyme that mediates Lys36 methylation in S. cerevisiae. Using the α-Me(Lys36)H3 antiserum, we screened S. cerevisiae whole-cell lysates prepared from strains with deletions of each of the SET domain-containing genes in S. cerevisiae. As shown in Fig. Fig.5C,5C, deletion of SET2 but not deletion of any other known SET domain-containing gene resulted in complete abolishment of Lys36 methylation, indicating that Set2 is likely to be the sole enzyme responsible for methylation at this site in budding yeast.

For comparison and as a control, a methyl-lysine 4-specific antiserum, α-Me(Lys4)H3, was also analyzed with this set of deletions. As previously observed (3, 3a), only deletion of SET1 results in abolishment of Lys4 methylation, demonstrating that this modification is mediated specifically by Set1 (Fig. (Fig.5C).5C). Taken together, these data suggest that Lys4 and Lys36 H3 methylation is catalyzed by Set1 and Set2, respectively. The putative site and substrate specificity of the other SET domain-containing proteins in S. cerevisiae (Set3 to Set7) are currently unknown.

To determine if the loss of H3 Lys36 methylation in the SET2 deletion strain could be restored by catalytically active forms of Set2, the Set2 expression construct previously used to rescue peak 1 HMT activity (see Fig. Fig.3)3) was assayed for the presence of Lys36 methylation. As shown in Fig. Fig.5D,5D, expression of Set2 but not the vector-only control or the two catalytically dead point mutations in Set2 (R195G and C201A; see Fig. Fig.4A)4A) restored H3 Lys36 methylation in this assay. These data strongly suggest that Set2 is responsible for bringing about Lys36 H3 methylation in vivo in S. cerevisiae.

Set2 functions as a transcriptional repressor in S. cerevisiae.

To our knowledge, little information is known about the biological role of Set2, although one published abstract suggests that Set2 may function as a transcriptional repressor (L. Lutfiyya, T. Hesman, and M. Johnston, abstract, Yeast 11:S220, 1995). To further investigate this possibility, an expression plasmid containing the DNA-binding domain of LexA fused to the C terminus of Set2 was constructed and transformed into S. cerevisiae along with a lacZ reporter construct driven by a heterologous CYC1 promoter containing a single copy of the LexA operator (lexAop). As a positive control for transcriptional repression, we also analyzed an Rpd3-LexA expression construct under identical assay conditions (43).

As shown in Fig. Fig.6A,6A, tethering of Set2-LexA showed a dramatic 20-fold repression of transcription, indicating that Set2 can act as a potent transcriptional repressor under these assay conditions. In comparison, Rpd3-LexA induced a 6.6-fold reduction in basal lacZ gene expression compared to the LexA DNA-binding domain alone. This result is comparable to previously published reports on the repressive effects of Rpd3-LexA using a similar lexAop-lacZ reporter construct (16). We note that in parallel experiments using an independent strain containing an integrated MFA2 promoter with a single copy of the lexA operator, similar levels of repression were observed by Set2-LexA (data not shown). To investigate the possibility that Set2 may activate transcription, we employed a strain that contained a GAL1 promoter containing a lexA operator fused to lacZ. Results showed that Set2-LexA, as well as Rpd3-LexA, exhibited no ability to activate transcription, while LexA-VP16 efficiently activated transcription (data not shown).

To investigate whether the point mutations previously engineered in the SET domain that abolish catalytic activity in vitro and in vivo (see Fig. Fig.4)4) affect the observed repression in our tethering experiments, Set2-LexA fusion constructs containing mutations at R195 and C201 [Set2(R195G)-LexA and Set2(C201A)-LexA] were generated and analyzed as described above. Results show a modest but significant (P < 0.05) reduction in the level of repression observed by Set2 when the SET domain mutants of Set2 were analyzed (both were reduced to ≈12-fold), suggesting that the HMT activity of Set2 plays a partial role in mediating transcriptional repression in this context (Fig. (Fig.6A).6A). Using an anti-LexA polyclonal antiserum, all LexA-containing proteins were found to be expressed at similar levels (Fig. (Fig.6B).6B). These results indicate that Set2 can act as a transcriptional repressor in this setting that functions, at least in part, through the catalytic activity of its SET domain (i.e., methylation of Lys36 in H3).

DISCUSSION

In this report, we have demonstrated that the S. cerevisiae protein Set2 is a nucleosomal H3 methyltransferase that is highly selective for Lys36, a lysine embedded in the more structured region of the H3 tail domain (21). We found that this site of methylation was highly conserved throughout eukaryotes in vivo and showed that Set2, via its SET domain, was responsible for Lys36 methylation in vitro and in vivo. In addition, we provide evidence that Set2 can function as a transcriptional repressor when tethered to a heterologous promoter in S. cerevisiae, a result consistent with its previous identification as a repressor for transcription (Lutfiyya et al., abstract). The observed repression appears to be partially mediated through the catalytic activity of the SET domain, indicating a role for Lys36 H3 methylation in downregulating gene expression, at least in this context.

Set2 and other SET domain-containing proteins in S. cerevisiae.

In S. cerevisiae, there are seven unique SET domain-containing proteins, the majority of which are not functionally characterized (34). Of these, Set1 and Set2 were the first identified and characterized as containing SET domains (20, 27; Lutfiyya et al., abstract). In our biochemical survey of S. cerevisiae whole-cell extracts, we identified two nucleosomal H3 HMT activities, one of which was shown to be Set2. As well, Set1 has recently been shown to be responsible for H3(Lys4) methylation in budding yeast (3; this report). Initially, we hypothesized that the minor HMT peak 2 (see Fig. Fig.1)1) would be catalyzed by Set1. However, deletion of Set1 does not result in a loss of either peak 1 or peak 2 HMT activity (data not shown), indicating that peak 2 represents a yet unidentified H3-specific, nucleosomal HMT. The site and substrate specificity of the remaining SET domain-containing proteins in S. cerevisiae are yet to be determined.

Set2 as a repressor of transcription.

Recent studies have revealed histone methylation as being an important regulator of chromatin function, and to date, these studies document histone methylation as playing both positive and negative roles in the process of gene transcription (14). The initial identification of Set2 was made on the basis of identifying proteins in a genetic screen that, when mutated, relieved basal repression of the GAL4 promoter (Lutfiyya et al., abstract). In agreement, our work shows that Set2 is a potent repressor of transcription when targeted to a heterologous promoter. While the observed repression is partially mediated by the activity of the SET domain, a significant amount of repression remains in our catalytically dead mutants.

Several possibilities may account for this discrepancy. First, since our tethering experiments were performed in WT strains, the remaining repression observed in the presence of the catalytically dead Set2-LexA mutants may be a result of endogenous Set2 activity. However, tethering experiments with these LexA constructs in a set2Δ strain revealed similar results (data not shown). Alternatively, this remaining repression might be mediated by Set2's ability to recruit additional proteins that aid in the repression process, a result that has been observed for other corepressors such as Rpd3 (5, 6). Similar to what we have observed with catalytically defective Set2 mutants, inactive forms of Rpd3 can also mediate transcriptional repression, and these defective Rpd3 proteins still recruit additional repressive factors (16). Recruitment of additional factors by Set2 might occur through its SET domain, as this domain has been shown to be a protein-protein interaction motif (15). Set2 also contains a WW (proline-rich) binding motif (see Fig. Fig.2C).2C). Whether this motif plays any role in the ability of Set2 to bind other proteins is not known.

In consideration of Set2 as a regulator of transcription, how Set2 is targeted remains an intriguing question. Size estimation of the Set2-mediated peak 1 activity indicates a native molecular mass of approximately 175 kDa (Fig. (Fig.2A),2A), suggesting that Set2 may function as either a homodimer or in association with another, yet unidentified factor(s). Given that Set2 shows no evidence for any kind of DNA-binding motif, it is likely that Set2 functions as a corepressor that is targeted through direct protein-protein interaction with other factors. In support, we find no evidence for the ability of Set2-Flag to coimmunoprecipitate Set2-LexA from whole-cell extracts from S. cerevisiae cells coexpressing Flag-tagged Set2 (see Fig. Fig.3)3) and Set2-LexA (see Fig. Fig.6)6) (data not shown).

Exclusive role for H3 lysine 36 methylation in gene silencing?

While sites of methylation have long been identified, only recently has the functional significance of these modifications begun to be elucidated. Based on our tethering experiments (Fig. (Fig.6),6), we suggest that Lys36 methylation, mediated by Set2, plays a role in mediating gene repression, at least under some circumstances. However, we do not rule out the possibility that this modification also may play a positive role for transcription as well. In support, H3(Lys36) methylation, like H3(Lys4) methylation (36), occurs in transcriptionally active macronuclei in Tetrahymena (data not shown), suggesting that this mark may function to potentiate transcription. Recent data on H3(Lys4) methylation suggest that this modification functions as part of a long-range, epigenetic marking system that serves to remodel large chromatin domains of chromatin into a state more permissive for gene transcription (reviewed in reference 42). Interestingly, the same H3(Lys4) methyl mark also plays a positive role in ribosomal DNA gene silencing (3, 3a). Thus, it seems likely that each specialized chromatin environment may influence gene regulation through chromatin modifications in different ways. The extent to which Lys36 H3 methylation is a targeted or more global phenomenon, influencing gene expression in a positive and/or negative fashion, remains an important issue for future investigations.

Acknowledgments

This research was supported by grants from the National Institutes of Health to C.D.A. (GM53512), D.F.H. (GM37537-14), and B.D.S. (GM20039). P.A.G. is the recipient of a Burroughs Wellcome Fund Career Award in Biomedical Sciences. S.D.B. is a Leukemia and Lymphoma Society Fellow. Z.-W.S. is supported by a postdoctoral cancer training grant from the University of Virginia Cancer Center.

We thank Karolin Luger (Colorado State University) for providing recombinant histones for these studies and Mike Hampsey (Robert Wood Johnson Medical School) for S. cerevisiae strains and DNA constructs. Also, we thank the research and development team at Upstate Biotechnology Inc. (Lake Placid, N.Y.) for assistance with the development of the H3 Lys36 methyl-specific antiserum. We thank Mary Bryk and Fred Winston (Harvard Medical School) as well as current Allis laboratory members, especially Craig Mizzen, Arthur Hsu, and Peter Chueng, for many helpful discussions and technical advice.

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