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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2002 Dec 10;99(Suppl 4):16438–16445. doi: 10.1073/pnas.182436399

Histone H3 lysine 4 methylation is mediated by Set1 and promotes maintenance of active chromatin states in fission yeast

Ken-ichi Noma 1, Shiv I S Grewal 1,*
PMCID: PMC139906  PMID: 12193658

Abstract

Methylation of histone H3 at lysine 4 (H3 Lys-4) or lysine 9 (H3 Lys-9) is known to define active and silent chromosomal domains respectively from fission yeast to humans. However, in budding yeast, H3 Lys-4 methylation is also necessary for silent chromatin assembly at telomeres and ribosomal DNA. Here we demonstrate that deletion of set1, which encodes a protein containing an RNA recognition motif at its amino terminus and a SET domain at the carboxy terminus, abolishes H3 Lys-4 methylation in fission yeast. Unlike in budding yeast, Set1-mediated H3 Lys-4 methylation is not required for heterochromatin assembly at the silent mating-type region and centromeres in fission yeast. Our analysis suggests that H3 Lys-4 methylation is a stable histone modification present throughout the cell cycle, including mitosis. The loss of H3 Lys-4 methylation in set1Δ cells is correlated with a decrease in histone H3 acetylation levels, suggesting a mechanistic link between H3 Lys-4 methylation and acetylation of the H3 tail. We suggest that methylation of H3 Lys-4 primarily acts in the maintenance of transcriptionally poised euchromatic domains, and that this modification is dispensable for heterochromatin formation in fission yeast, which instead utilizes H3 Lys-9 methylation.

Dynamic changes in chromatin structure are directly influenced by the posttranslational modification of histones. Specific amino acids on histone amino-terminal tails that extend outward from nucleosome core particle are the targets of a number of modifications including acetylation, phosphorylation, ubiquitylation, ADP ribosylation, and methylation (1–5). The presence of a specific pattern of histone modifications has been linked to various chromosomal processes, such as the maintenance of gene expression patterns during development, recombination, chromosome condensation, and the proper segregation of chromosomes during mitosis. For example, hyperacetylated regions of chromatin contain active transcription units, whereas hypoacetylated chromatin is transcriptionally silent (6).

How histone modifications participate in the modulation of chromatin structure is not fully understood. It is believed that different combinations of histone modifications can determine the binding affinities of histone-interacting proteins whose chromosomal associations lead to discrete downstream events (2, 7). This is best illustrated by recent studies showing that modifications of the H3 tail by deacetylase and methyltransferase activities likely act in concert to establish the “histone code” essential for heterochromatin assembly (3, 4, 8). The chromodomains of heterochromatin proteins Swi6 and HP1 from fission yeast and Drosophila, respectively, are specific interaction motifs for the histone H3 amino-terminal tail modified by methylation on lysine 9 (9–11), and localization of these proteins to heterochromatic loci depends on H3 Lys-9 methylation (8, 12). Similarly, the bromodomain of many transcriptional coactivators binds specifically to the acetylated lysine residues on histone tails (13). Applying this general concept to other chromatin modulators, it is likely that unique combinations of histone modifications serve as marks for the recruitment of different chromatin proteins or protein complexes to initiate the formation of defined chromosomal subdomains with distinct functions.

The methylation of H3 and H4 tails can occur on both arginine and lysine residues, and plays a critical role in transcriptional regulation (3, 5). A specific class of methyltransferases including CARM1 and PRMT1, which act as transcriptional coactivators, catalyze histone arginine methylation (14–16). However, enzymes that contain an evolutionary conserved SET domain, a 130-residue motif originally identified in Su(var)3–9, Enhancer-of-zeste and Trithorax proteins in Drosophila (17), are implicated in the lysine methylation of histones (5). The founding members of this class of histone methyltransferases are mammalian SUV39H1 and its fission yeast homolog Clr4, which specifically methylate H3 lysine 9 (H3 Lys-9) at heterochromatic loci (8, 18). Recently, several SET domain-containing proteins, such as G9a, SETDB1, and ESET, have been shown to methylate H3 Lys-9 in mammals (19–21).

In contrast to the H3 Lys-9 methylation that defines silent chromosomal domains, H3 Lys-4 methylation is specific to transcriptionally poised euchromatic regions in fission yeast, Tetrahymena, chicken, and mammals (22–26). Moreover, high-resolution mapping using chromatin immunoprecipitation (ChIP) has shown that distinct patterns of histone methylation marking heterochromatic and euchromatic domains are separated by boundary elements that protect against spreading of repressive chromatin into neighboring areas (22, 23). H3 Lys-4 is methylated by SET domain containing proteins—Set1 in Saccharomyces cerevisiae (27–29) and Set7/Set9 in mammals (30, 31). Although the precise function of H3 Lys-4 methylation is not known, it has been hypothesized to facilitate transcription by serving as a mark to recruit necessary transcription machinery components, or alternatively to protect euchromatic regions from the repressive effects of neighboring silent chromatin complexes (22, 23).

Interestingly, the deletion of SET1 in S. cerevisiae leads to defects in silencing at telomeres, ribosomal DNA (rDNA), and the mating-type region (27, 32, 33). It has been suggested that H3 Lys-4 methylation in the context of other histone modifications might have a dual function both in transcriptional activation and silencing (27, 33). However, it remains to be investigated whether H3 Lys-4 methylation by Set1 homologs is also required for silent chromatin assembly in other species.

In this study, we report identification and structural features of at least nine SET domain proteins present in the fission yeast genome. We show that one of these SET domain proteins that we named Set1 appears to be the exclusive H3 Lys-4-specific methyltransferase. Unlike in S. cerevisiae, Set1 is not required for heterochromatin assembly in fission yeast, consistent with our previous results showing that H3 Lys-4 methylation is specific to euchromatic regions. H3 Lys-4-methylaton is present throughout the cell cycle and important for the upkeep of transcriptionally poised domains in euchromatic regions, perhaps through the maintenance of histone acetylation levels.

Materials and Methods

Sequence Analysis.

Database searches were performed with blastp. Multiple amino acid sequences were aligned by using clustalw, Version 1.7. A phylogenetic tree was created by the neighbor-joining method based on the amino acid sequence alignment. The domains were characterized by using the motifscan program against the prosite database. The resulting dendrogram showing the relationship between different SET domain proteins is shown in Fig. 1A, and structural features of different SET domain proteins in fission yeast are shown in Fig. 1B.

Figure 1.

Figure 1

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SET domain proteins in Sch. pombe genome. (A) Phylogenetic tree of the SET domain proteins. The tree was constructed by the neighbor-joining method based on the amino acid sequences in the SET domains. The scale bar equals a distance of 0.05 aa. The SpSet (Sch. pombe SET) members (GenBank accession nos.: SpSet1, AL049728; SpSet2, Z99164; SpSet3, Z70043; SpSet5, AL031540; SpSet6, AL032684; SpSet7, AL049609; SpSet8, Z99568; SpSet9, AL132870) were identified by blast using previously identified SET domains as query. Abbreviations: Hs, Homo sapiens; m, Mus musculus. (B) Schematic representation of Sch. pombe SET domain proteins. The length of each protein (in aa) is noted on the right. Conserved domains are indicated as follows: RRM, RNA recognition motif; Chromo, Chromodomain; PHD, PHD finger; Cys-rich, Cysteine-rich domain; Ser-rich, Serine-rich domain.

Strains.

The genotypes of the Schizosaccharomyces pombe strains used in this study are listed in Table 1. The set1Δ strain was constructed by a PCR based method as described (34). Deletion was confirmed by PCR and Southern analysis. A strain containing deletion of a part of the set1 ORF encoding RNA recognition motif (RRM) was constructed as follows: DNA fragments from upstream (0.9 kb) and downstream (2.6 kb) of RRM encoding region of set1 were amplified by PCR with PfuTurbo DNA polymerase (Stratagene) and cloned in frame into pCRII-TOPO (Invitrogen) to construct set1 ORF minus RRM encoding region (set1ΔRRM). The resulting 3.5-kb fragment was gel-purified and used for transformation of SPK10 strain carrying set1Δ∷kanMX6 allele. Transformants were screened for G418 sensitivity, and colonies carrying set1ΔRRM were confirmed by using PCR analysis. Standard genetic crosses were used to construct all other strains.

Table 1.

Sch. pombe strains used in this study

Strain ura4+ insertion Genotype
FY498 imr1R∷ura4+ h+ leu1-32 ura4DS/E ade6-210
FY648 otr1R∷ura4+ h+ leu1-32 ura4DS/E ade6-210
PG925 mat3M∷ura4+ h90 leu1-32 ura4D18 ade6-210 clr3-735
SP1464 KΔ∷ura4+ h90 leu1-32 ura4D18 ade6-210 clr6-1
SPG1236 Kint2∷ura4+ h90 leu1-32 ura4DS/E ade6-216 his2
SPK10 Kint2∷ura4+ h90 leu1-32 ura4DS/E ade6-216 his2 set1Δ∷kanMX6+
SPK108 imr1R∷ura4+ h+ leu1-32 ura4DS/E ade6-216 set1Δ∷kanMX6+
SPK111 otr1R∷ura4+ h+ leu1-32 ura4DS/E ade6-210 set1Δ∷kanMX6+
SPK121 Kint2∷ura4+ h90 leu1-32 ura4DS/E ade6-216 his2 set1ΔRRM
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Iodine Staining Assay.

Efficiency of mating-type switching was analyzed by the iodine-staining assay. Individual colonies were replicated onto sporulation (PMA+) medium and then grown for 3 days at 26°C before being exposed to iodine vapors. The dark staining indicates efficient mating-type switching, which requires heterochromatin-mediated chromatin organization at the mating-type region (35). Defects in heterochromatin assembly at the mating-type region results in inefficient switching, causing a decrease in iodine staining.

Western Analysis of Histones.

For isolation of bulk histones, fission yeast cells were grown to mid-log phase. Cells (5 × 108) were washed with 10 ml of NIB buffer (0.25 M sucrose/60 mM KCl/15 mM NaCl/5 mM MgCl2/1 mM CaCl2/15 mM Pipes, pH 6.8/0.8% Triton X-100) and resuspended in 500 μl of NIB buffer containing 10 ng/μl TSA, 2 mM ZnSO4, Complete protease inhibitor mixture (Roche, 1 tablet per 10 ml), and 1 mM PMSF. Cells were disrupted by acid-washed glass beads (425–600 μM) using a minibeadsbeater (Biospec) for 4–5 min. Cell extracts were centrifuged at 11,000 × g for 10 min. The pellets were resuspended in 0.4 M H2SO4 and incubated on ice for 1 h with occasional mixing. The supernatant was collected by centrifugation at 8,000 × g for 5 min. The H2SO4 extraction was repeated. Pooled supernatants were trichloroacetic acid precipitated and pellets were washed twice in 500 μl of cold acetone, air-dried and resuspended in 10 mM Tris⋅HCl, pH 8.0. Bulk histone samples were kept in −70°C freezer until use. Ten micrograms of crude histone samples were resolved on an SDS/18% PAGE, transferred to a poly(vinylidene difluoride) membrane, and probed with site-specific acetyl- or methyl-histone antibodies. Antibodies to H3 Lys-4-methyl, H3 Lys-9-acetyl, and H3 Lys-14-acetyl were purchased from Upstate Biotechnology, whereas antibodies specific to acetylated Lys-5, Lys-8, Lys-12, or Lys-16 of histone H4 were purchased from Serotec. The band intensities were quantified with NIH IMAGE 1.62 software.

Immunofluorescence Analysis.

Cells were grown to mid-log phase in yeast-extract adenine (YEA) medium. An equal volume of YEA + 2.4 M sorbitol was added and the culture was incubated further at 18°C for 5 min. For using α-H3 Lys-4-methyl, cells were fixed by adding paraformaldehyde to a final concentration of 1.7% and incubated at 18°C for 45 min. For staining by α-H3 Ser-10-phospho, cells were fixed in 3.0% paraformaldehyde for 30 min. Fixed cells were treated with Zymolyase to permeabilize the cell wall and then incubated overnight with primary antibodies, such as mouse α-tubulin TAT1 (1:150 dilution) or α-Nop1 (1:1,000 dilution) and rabbit α-H3 Lys-4-methyl (1:1,500) or α-H3 Ser-10-phospho (1:500). After extensive washing, cells were incubated for 6–8 h with Alexa Fluor 594 anti-rabbit IgG and Oregon Green 488 anti-mouse IgG (Molecular Probes) at a 1:2,000 dilution. After washing, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), mounted in Vectashield mounting medium (Vector Laboratories), and analyzed by a Zeiss Axioplan 2 fluorescence microscope.

ChIP.

ChIP analysis was performed as described (36, 37). Fission yeast cells grown at 32°C in YEA (5 × 108 cells at 1 × 107 cells per ml for each reaction) were shifted to 18°C for 2 h before 30-min fixation in 3% paraformaldehyde. Soluble chromatin fractions prepared from fixed cells were sheared to ≈0.5- to 0.8-kb DNA fragments by sonication before immunoprecipitating by using antibodies to H3 Lys-4-methyl, H3 Lys-9-methyl, H3 Lys-14-acetyl, and Swi6. DNA fragments recovered from immunoprecipitated chromatin fractions or from whole cell crude extracts were subjected to PCR analyses (94°C for 30 s , 55°C for 30 s, 72°C for 1 min, 30 cycles). PCR products were labeled by including 0.25 μl of [α-32P]deoxycytidine triphosphate (10 mCi/ml; 1 Ci = 37 GBq) in each reaction. PCR products were separated on a 4% polyacrylamide gel, and band intensities were quantified by using a Fuji PhosphoImager.

Results

SET Domain Proteins in Sch. pombe and Their Structural Features.

A database search of the Sch. pombe genome (http://www.sanger.ac.uk/Projects/S_pombe/) was performed to identify the total number of SET domain proteins. The Sch. pombe genome has been sequenced, and contains 4,824 genes (38). A blast search with the SET domain of the Clr4 histone methyltransferase revealed that at least nine SET domain proteins, including Clr4, reside in the fission yeast genome (Fig. 1A). All of the Sch. pombe SET domain proteins contain a conserved NHSC motif, which, when mutated, has been shown to abolish histone methyltransferase activity (18). SpSet1, SpSet2, and SpSet3 were named after their S. cerevisiae counterparts based on homology. However, Clr4, SpSet5, SpSet6, SpSet7, SpSet8, and SpSet9 did not share significant similarities to S. cerevisiae proteins outside of their SET domain. The absence of a Clr4 homolog in S. cerevisiae is consistent with the fact that bulk histones isolated from budding yeast cells lack detectable levels of H3 Lys-9 methylation (24, 27).

We next carried out phylogenetic analysis to assess the relationships between different SET domain proteins (Fig. 1A). The SET domain sequences of Sch. pombe proteins and previously characterized proteins from other species were aligned together and a phylogenetic tree was constructed. Based on the dendrogram (Fig. 1A), Sch. pombe SET domain proteins were not highly related to one another. As expected, SpSet1 and Clr4 proteins cluster with the previously described Set1 and SUV39 family of histone methyltransferases that have the capacity to methylate H3 Lys-4 and H3 Lys-9, respectively (5). The Set1 proteins from budding and fission yeast share 26% identity throughout their length and 63% in the SET domain region. Our analysis suggests that SpSet1 is more closely related to Set1 in Homo sapiens than it is to Set1 in S. cerevisiae. Another SET domain protein, SpSet2, is closely related to a H3 Lys-36-specific methyltransferase from S. cerevisiae (39).

Careful examination of the SET domains in Sch. pombe proteins revealed distinct structural features. As reported previously, the SET domain in Clr4 is surrounded by two cysteine-rich regions, referred to as preSET and postSET, that are essential for its catalytic activity (8). The preSET and postSET domains are also present in SpSet2, but only the postSET domain is present in SpSet1, SpSet5, SpSet6, and SpSet9 (Fig. 1B). SpSet1 contains a highly conserved 160-aa motif called n-SET at the preSET location. The SET domains of SpSet3, SpSet7, and SpSet8 do not have pre- or postSET motifs. It is possible that differences in structural features surrounding the SET domains might account for the altered substrate specificity of these proteins.

Further analysis using the motifscan program revealed that in addition to the presence of a chromodomain in Clr4, as reported (40), SpSet1 and SpSet3 contain an RRM and PHD finger at their amino-terminal regions, respectively. Both the PHD finger that is involved in protein–protein interactions, and the RRM, which is known to interact with both RNA and proteins, are conserved motifs shown to be present in subunits of chromatin-modifying activities (41, 42).

Sch. pombe Set1 Is Required for H3 Lys-4 Methylation in Vivo.

As mentioned above, Set1 mediates H3 Lys-4 methylation in S. cerevisiae (27–29). Considering that Set1 proteins from budding and fission yeasts share considerable homology within and outside of their SET domains, it was possible that SpSet1 might also be involved in H3 Lys-4 methylation. To test this possibility, we constructed a strain containing a complete deletion of set1 gene, replacing the entire ORF with the KANMX6 gene. The resulting set1Δ mutant was viable, suggesting that SpSet1 is dispensable for cell growth.

To study the biological effects of set1Δ on H3 Lys-4 methylation, we performed immunofluorescence analysis using an antiserum specific to methyllysine 4 of histone H3 (22, 27). In wild-type cells, immunofluorescence signal corresponding to H3 Lys-4-methyl was preferentially enriched at the chromatin in the DAPI-stained areas but seemed to be excluded from the nucleolus containing rDNA repeats, as indicated by the staining of nucleolar marker protein Nop1 (Fig. 2A; ref. 43). In comparison to a high level of H3 Lys-4 methylation in the nuclei of wild-type cells, strikingly, methylation of histone H3 at lysine 4 was completely abolished in set1Δ cells (Fig. 2C), suggesting that SpSet1 is responsible for H3 Lys-4 methylation. This result was further confirmed by Western analysis of bulk histones prepared from wild-type and set1Δ cells by using H3 Lys-4-methyl-specific antibodies. Histones prepared from wild-type cells exhibit high levels of methylated H3 Lys-4, but we could not detect any signal in the set1Δ cells (Fig. 2B). These analyses suggest that Set1 mediates H3 Lys-4 methyaltion in Sch. pombe. Although it remains a possibility that low levels of H3 Lys-4 methylation exist in the nucleolus, histone H3 methylated at lysine 4 is clearly not enriched in the nucleolar compartment.

Figure 2.

Figure 2

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Deletion of set1 abolishes H3 Lys-4 methylation. (A) Wild-type cells (SPG1236) were stained with anti-H3 Lys-4-methyl (H3K4Me) antibody (red), anti-Nop1 antibody (green), and DAPI (blue). (B) Western blot with H3K4Me antibody against crude bulk histones prepared from wild-type (SPG1236) and set1Δ (SPK10) strains. Identical samples were examined in parallel by Coomassie staining to show histone loading. (C) Wild-type and set1Δ (SPK10) cells were stained with anti-H3K4Me and DAPI. (D) Comparison in growth rate of wild-type (SPG1236) and set1Δ (SPK10) strain. Wild-type and mutant strains were grown in YEA-rich medium at 30°C. Cell numbers at fixed time points are plotted on a semilogarithmic graph. (E) Temperature sensitivity of set1Δ strain. The strains previously grown under permissive growth conditions (30°C) were replicated onto YEA plates and incubated overnight at 30°C or 37°C.

Previous studies in S. cerevisiae have suggested that H3 Lys-4 methylation is important for normal growth, and that mutations in SET1 can result in a number of phenotypes including morphological abnormalities, perturbed DNA distribution, growth and sporulation defects (27, 32). We therefore investigated whether set1Δ cells display similar phenotypes in Sch. pombe. We noticed that colonies formed by set1Δ mutants are smaller in size when compared with their wild-type counterparts (data not shown). Furthermore, set1Δ cells have a slightly longer doubling time and exhibit temperature-sensitive growth defects (Fig. 2 D and E). However, deletion of Sch. pombe set1 did not cause any obvious morphological abnormalities, sporulation defects, or abnormal DAPI staining patterns.

RRM of SpSet1 is Necessary for Its Role in H3 Lys-4 Methylation.

As described above, the amino terminus of the SpSet1 contains a canonical RNA binding domain called RRM (Fig. 1B). Interestingly, this motif is also present in orthologs of SpSet1 in S. cerevisiae, Caenorhabditis elegans, Drosophila, and humans. We sought to investigate whether the RRM domain is essential for SpSet1-mediated H3 Lys-4 methylation. For this purpose, we constructed a strain in which a small part of the set1 ORF, encoding the RRM, was deleted at its endogenous chromosomal location. The expression of set1ΔRRM is under the control of the native set1 regulatory elements, so as to achieve wild-type levels of expression. Interestingly, Western analysis of bulk histones revealed that H3 Lys-4 methylation is severely defective in the set1ΔRRM strain as compared with wild-type (Fig. 3). Moreover, set1ΔRRM strain showed temperature-sensitive growth defects (data not shown). Although a possibility remains that deletion of RRM affects steady-state Set1 levels in the cells, we suggest that the RRM domain of SpSet1 might be required for its role in H3 Lys-4 methylation.

Figure 3.

Figure 3

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RRM of SpSet1 is required for its role in H3 Lys-4 methylation. (A) Schematic representation of the Set1 and Set1ΔRRM. (B) Western blot with H3K4Me antibody against crude bulk histones prepared from wild-type (SPG1236), set1Δ (SPK10), and set1ΔRRM (SPK121) strains. Coomassie brilliant blue (CBB) staining is shown as loading controls.

Deletion of set1 Does Not Affect Silencing at the Mating-Type Region and Centromeres.

Modifications of histone tails are known to play a critical role in heterochromatin assembly through their role in dictating the interactions between nucleosome arrays and non-histone chromatin proteins (44). Set1-mediated H3 Lys-4 methylation has been shown to be required for transcriptional silencing of the silent mating-type loci, telomeres, and rDNA in S. cerevisiae. To investigate whether set1 is also required for transcriptional silencing in Sch. pombe, we combined set1Δ with a ura4+ marker gene inserted at either the silent mating-type region (Kint2∷ura4+) or at two different sites within cen1, one each at the outer (otr1R∷ura4+) and inner (imr1R∷ura4+) centromeric repeats that flank the central (cnt) domain (Fig. 4; ref. 45). Previous studies have shown that marker genes inserted within or adjacent to these heterochromatic locations are subject to transcriptional repression, as a consequence of repressive chromatin complexes spreading into the marker genes (46). Wild-type yeast cells carrying repressed ura4+ at a heterochromatic site cannot form colonies on medium lacking uracil (URA−) but grow efficiently on a counterselective medium containing 5′-fluoroorotic acid (FOA). However, cells defective in silencing grow on URA− medium and are FOA sensitive. Dilution analysis of the wild-type and set1Δ strains carrying ura4+ marker gene at the silent mating-type region or at the centromeric locations revealed that SpSet1 is not required for silencing at these loci, as indicated by comparable growth of wild-type and set1Δ cells on URA− and FOA media (Fig. 4 B and C).

Figure 4.

Figure 4

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Effects of set1 deletion on silencing and heterochromatin assembly. (A) Iodine-staining assay. Wild-type and set1Δ colonies were sporulated on PMA+ medium at 25°C and exposed to iodine vapors before photography. (B and C) Deletion of set1 does not affect silencing at the mating-type region and centromere. The strains that contain ura4+ genes inserted at the mating-type locus (Kint2∷ura4+) or centromere region of chromosome I (imr1R∷ura4+ and otr1R∷ura4+) were used for ChIP assays with antibodies to H3 Lys-4-methyl (K4), H3 Lys-9-methyl (K9), or Swi6 protein. DNA from ChIP or WCE (whole cell crude extract) was analyzed by a competitive PCR strategy, whereby one set of primers amplifies different-sized products from the ura4+ marker gene and the control ura4DS/E minigene at the endogenous euchromatic location. The ratios of ura4+ and control ura4DS/E signals present in ChIP and WCE were used to calculate relative enrichment, shown beneath each lane. NE indicates no enrichment observed. The ura4+ expression levels at the mating-type and cen1 region were evaluated by dilution analysis. Cells were suspended in water, and 10-fold serial dilutions were spotted onto nonselective (N/S), counterselective FOA, AA-URA medium, and grown 3 days before being photographed.

We also tested whether set1Δ affects expression of the KΔ∷ura4+ marker gene. The KΔ∷ura4+ cells containing a substitution of part the interval between silent mating-type loci with the ura4+ marker gene exhibit variegated ura4+ expression (37). ura4-off and ura4-on epigenetic states are mitotically metastable. We used fluctuation analysis to measure the effect of set1Δ on stability of the epigenetic states. The set1Δ had a subtle effect on the ura4-off to ura4-on transition (7.9 × 10−4 per cell division), as compared with wild-type cells (8.4 × 10−4 per cell division). Moreover, we observed that set1Δ caused a slight decrease (9.6 × 10−4 to 4.6 × 10−4 per cell division) in ura4-on to ura4-off conversion. This decrease in the “on” to “off” state conversion in set1Δ background could be caused by changes in the levels of trans-acting factors critical for establishment of the silenced state.

In addition to silencing, the efficiency of mating-type switching is also regulated by formation of a heterochromatic structure at the mat locus. Mutations that affect silencing and heterochromatin assembly at the mating-type region adversely affect mating-type interconversion (47, 48). We monitored the efficiency of mating-type switching in wild-type and set1Δ strains at the colony level by iodine-staining as described in the Materials and Methods section. We found that set1Δ has no effect on the efficiency of mating-type switching (Fig. 4A), consistent with our results that expression of the marker gene inserted at the silent mating-type region is not affected.

SpSet1 Is Dispensable for Heterochromatin Assembly.

Silencing at the mating-type region and centromeres depends on Swi6 protein, which is recruited to these loci through its binding to methylated H3 Lys-9 (46). We therefore explored whether set1Δ affects H3 Lys-9 methylation and/or Swi6 at heterochromatic loci. ChIP assays with antibodies to Swi6 or H3 Lys-9-methyl were used. DNA recovered from immunoprecipitated chromatin fractions was quantitated by using competitive PCR, whereby one primer pair amplifies 694-bp and 426-bp products from full-length ura4+ inserted at the heterochromatic location and a mini-ura4 (ura4DS/E) at its endogenous euchromatic location. Our analysis revealed that the levels of H3Lys9 methylation and Swi6 at the silent mating-type region (Kint2∷ura4+) and centromeric repeats (imr1R∷ura4+ and otr1R∷ura4+) of set1Δ cells were comparable to their wild-type counterparts (Fig. 4 B and C). As expected, set1Δ abolished the preferential enrichment of H3 Lys-4 methylation at the euchromatic ura4DS/E locus. Taken together, the results presented above suggest that Set1-mediated H3 Lys-4 methylation is not required for heterochromatin assembly in Sch. pombe.

H3 Lys-4 Methylation Is Present Throughout the Cell Cycle.

In comparison to the acetylation of histones, lysine methylation is believed to be a relatively stable histone modification, which might serve as an epigenetic imprint for the long-term maintenance of chromatin states. If H3 Lys-4 methylation indeed serves as a molecular bookmark for inheritance of the active chromatin state, it is likely to be present throughout the cell cycle, even during mitosis. To address this issue, we performed immunofluorescence with antibodies to methylated H3 Lys-4 and tubulin used to visualize microtubules. As a control, we also studied H3 Ser-10 phosphorylation, which correlates with chromosome condensation during mitosis (49). At the G2/M boundary, H3 Ser-10 phosphorylation was mainly localized to one or two discrete foci. However, Ser-10 phosphorylation spread throughout chromosomes by metaphase, the intensity of the signal diminished as cells enter anaphase, and almost all staining had disappeared in G1/S cells (Fig. 5). In contrast to H3 Ser-10 phosphorylation, we found that H3 Lys-4 methylation levels remained unchanged throughout the cell cycle, including mitosis when chromosomes are highly condensed. Although changes in Lys-4 methylation at individual loci cannot be ruled out, H3 methylated at Lys-4 can be detected during different stages of the cell cycle. Because covalent modification by one enzyme can positively or negatively influence the efficiency of other enzymes responsible for modifying residues on the same histone tail, we also tested the effects of set1Δ on H3 Ser-10 phosphorylation. As shown in Fig. 5, the loss of H3 Lys-4 methylation in set1Δ strains did not affect H3 Ser-10 phosphorylation during mitosis.

Figure 5.

Figure 5

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H3 Lys-4 methylation is present throughout the cell cycle. (A) Wild-type cells were stained with anti-H3K4Me (red), anti-tubulin TAT-1 antibody (green), and DAPI (blue). For each cell, the corresponding panels are placed below each other. (B) Wild-type and set1Δ cells were stained with antibodies to phosphorylated H3 Ser-10 (H3S10P).

Interplay Between H3 Lys-4 Methylation and Histone Acetylation in Vivo.

It has been shown that Lys-4 methylation of H3 is preferentially associated with H3 acetylation in S. cerevisiae, chicken, and HeLa cells (23, 24). Here we examined whether SpSet1-mediated H3 Lys-4 methylation affects histone acetylation in vivo. Bulk histones prepared from wild-type and set1Δ were subjected to Western blot analysis with acetylation-site-specific H3 or H4 antibodies (Fig. 6). Interestingly, we found that the acetylation levels of histone tails, in particular of H3 Lys-9 and H3 Lys-14, were significantly decreased in set1Δ cells when compared with wild-type cells (Fig. 6). The set1Δ also results in a subtle but consistent decrease in H4 Lys-5 and H4 Lys-12 acetylation. We also investigated the possible effects of mutations in the histone deacetylases clr3 and clr6 on H3 Lys-4 methylation in bulk histones in vivo. As shown recently, clr3 specifically affects H3 Lys-14 acetylation, whereas mutation in clr6 results in elevated acetylation levels at all residues tested on the histone H3 and H4 tails (Fig. 6; ref. 50). Although H3 Lys-4 was slightly more methylated in clr6 mutant, mutation in clr3 had no effect on Lys-4 methylation. These data suggest that H3 Lys-4 methylation might help promote acetylation of histones in the transcriptionally poised regions of the chromosomes.

Figure 6.

Figure 6

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set1Δ strain exhibits decreased levels of acetylation at lysine residues in H3 tail. (A) Bulk histones were prepared from wild-type, set1Δ, clr3–735, and clr6-1 strains. Ten micrograms of crude histones were separated by SDS/PAGE and subjected to Western analysis with antibodies specific for H3K4me, or histone H3 or H4 acetylated at the indicated lysine residues. Coomassie staining (CBB) is shown as the loading control. Similar results were obtained in at least three independent experiments. (B) The intensity of the bands shown in A, quantified by using NIH image software, is summarized.

We also analyzed the effects of H3 Lys-4 methylation on H3 acetylation levels by ChIP assays. The loss of H3 Lys-4 methylation in set1Δ cells causes 50–60% reduction in H3 Lys-14 acetylation levels at the constitutively expressed loci ura4, act1, and ade6, as compared with wild-type background cells (Fig. 7). Consistent with decreased histone acetylation, we also observed that set1Δ causes subtle changes in ade6 expression. Because H3 Lys-4 methylation seems to globally affect active chromatin regions, changes in ade6 expression could not be quantified using Northern analysis because of the lack of appropriate controls. However, we observed that set1Δ cells carrying the ade6-216 allele at its endogenous chromosomal location formed deep red colonies on adenine-limiting medium, as compared with the pink colonies formed by their wild-type counterparts (Fig. 7). This phenotype indicating a decrease in ade6 expression consistently segregated with the set1Δ in more than 30 tetrads. Taken together, these analyses suggest that H3 Lys-4 methylation might be involved in the maintenance of active chromatin configurations, presumably by facilitating the acetylation of histones.

Figure 7.

Figure 7

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set1Δ results in a decrease of H3 Lys-14 acetylation levels at genes. (A) ChIP analysis with antibodies to acetylated H3 Lys-14 (H3K14Ac). DNA fragments from the immunoprecipitated (IP) fraction and whole cell crude extract (W) were analyzed by using multiplex PCR. Relative enrichment of DNA fragments corresponding to the coding regions of ura4, act1, or ade6 was examined. A DNA fragment from the silent mating-type region (K-region), which is known to lack H3 Lys-4-methyl and H3 Lys-14-acetyl modifications, was used as a control to normalize and calculate the relative enrichment of ura4, act1, and ade6 sequences in IPed chromatin fractions. (B) Analysis of ade6-216 phenotype. Wild-type and set1Δ cells were streaked onto adenine-limiting yeast extract medium and incubated at 30°C for 2–3 days. Deep red or pink color of colonies indicates ade6-216 expression states. Colonies of set1Δ strains were deeper red than wild type, indicating decrease in ade6 expression in mutant cells.

Discussion

Epigenetic control of higher-order chromatin assembly has been linked to the posttranslational covalent modifications of the histones tails. It has been formally suggested that distinct modifications on one or more of the histone tails act sequentially or in combination to form a histone code that is recognized by other chromatin-associated proteins (2, 7). Although histone-modifying enzymes such as acetyltransferases and deacetylases have been identified and characterized from a number of organisms, the factors that regulate methylation of histones are only now being discovered. Recent studies have identified a novel class of protein methyltransferases defined by an evolutionarily well-conserved structure, the SET domain (5, 18).

In this paper, we report the structural features of at least nine SET domain proteins present in fission yeast. The data presented demonstrate that a highly conserved SET domain protein named SpSet1 likely catalyzes the H3 Lys-4 methylation present at euchromatic regions. Based on genetic and biochemical studies, the SET domain and its flanking pre- and postSET domains are required for catalytic activity of SUV39H1 and Clr4 methyltransferases (8, 18). However, recent studies argue against the general requirement of the pre- and postSET domains for enzymatic activity. For example, human Set7/Set9 contains a SET domain but is devoid of the pre- and postSET motifs, and can efficiently methylate H3 Lys-4 in vitro and in vivo (30, 31), though it is possible that other sequences surrounding the SET domain might promote methyltransferase activity. The general consensus emerging is that the SET domain constitutes the catalytic motif, whereas flanking sequences might facilitate folding of the histone tails, providing specificity for a particular lysine residue. In this regard, the highly conserved n-SET motif and cysteine rich sequences flanking the SET domain in Set1 orthologs are likely to be critical for their specificity to H3 Lys-4. (Fig. 1; ref. 28). Based on conservation of the SET domain in yet uncharacterized SET domain proteins in fission yeast, it is probable that these proteins also act as methyltransferases.

In many cases, SET domain proteins also contain other conserved motifs such as the chromodomain, PHD finger, and RRM domain. For example, the SUV39 family of proteins including Clr4 is known to contain a chromodomain at their amino terminus (40). Although the SET domain of Clr4 and its surrounding sequences are sufficient for methyltransferase activity in vitro, both SET domains and chromodomains are required in vivo (8). We found that the RRM domain of Sch. pombe Set1 is required for its role in H3 Lys-4 methylation in vivo. The precise function of the RRM is not known, but it is possible that RRMs and chromodomains have related functions. These domains might help promote chromosomal targeting of their respective proteins, either through protein–protein interactions or through their binding to RNA. Supporting the possible role for RNAs in chromatin assembly, our recent work suggests that RNA interference (RNAi) mechanisms, through which small RNAs silence cognate genes, might be required for the targeting of histone-modifying activities to specific chromosomal domains in fission yeast. Specifically, RNAi machinery is essential for histone deacetylation and Clr4-mediated H3 Lys-9 methylation at centromeric repeats (I. Hall and S.I.S.G., unpublished data). Considering that certain chromodomains act as an RNA interaction module, it can be imagined that the binding of chromo- and/or RRM domains to RNA might guide the histone-modifying activities to homologous genomic sequences. In this scenario, RNA might provide specificity for the chromosomal targeting of these enzymes. Of course, it remains a possibility that chromodomains and RRM domains associated with these SET domain proteins are protein–protein interaction motifs. Future studies are necessary to address these possibilities.

Recent work suggests that H3 Lys-4 methylation can perform dual functions in S. cerevisiae. Interestingly, Set1-mediated H3 Lys-4 methylation at rDNA and telomeres in S. cerevisiae is required for transcriptional silencing (27, 32, 33). It has been suggested that H3 Lys-4 methylation in combination with other histone modifications could have a negative or positive effect on transcription at different chromosomal locations (27, 33). In this regard, our analysis suggests that Sch. pombe Set1 is not required for transcriptional silencing and heterochromatin assembly at centromeres or the silent mating-type interval. The differences in species of H3 Lys-4 that are mono-, di-, or tri-methylated might explain these seemingly contradictory results. A more likely explanation is that the histone code for silent chromatin assembly in S. cerevisiae and Sch. pombe is fundamentally different. H3 Lys-4 methylation is used in the process of silent chromatin assembly in budding yeast, whereas H3 Lys-9 methylation is used for formation of repressive chromatin structures in fission yeast and higher eukaryotes. Consistent with this idea, H3 Lys-9 methylation has not been detected in S. cerevisiae, and homologs for neither the H3 Lys-9 methyltransferase nor the Swi6/HP1 protein that recognizes this modification are present (27).

The mechanism by which H3 Lys-4 methylation modulates chromatin structure is not clear. The budding yeast Set1 is found in a complex with homologs of the Drosophila protein Ash2 and Trithorax, which are essential for the stable maintenance of active gene expression states during development (28, 29). Furthermore, recent studies have shown that H3 Lys-4 methylation is preferentially associated with euchromatic regions and is correlated with H3 acetylation (22–25). Our analysis suggests that H3 Lys-4 methylation and acetylation at the H3 tail interact in cis at euchromatic regions in vivo. We found that set1Δ results in a decrease in H3 acetylation levels. Furthermore, we also observed that a mutation in the Clr6 histone deacetylase, which displays broad specificity to lysine residues at H3 and H4 tails (ref. 50 and this study), results in a slight increase in H3 Lys-4 methylation. Based on these results, it is possible that H3 Lys-4 methylation either plays a facilitatory role in histone acetylation or it protects transcriptionally active regions from the effects of repressive chromatin remodelling activities such as histone deacetylases, or both. Supporting our in vivo analysis, it has been shown recently that H3 Lys-4 methylation facilitates subsequent acetylation of the histone tail by acetyltransferases in vitro (30). Furthermore, methylation of H3 Lys-4 interferes with interactions between the NuRD histone deacetylase and H3 tail, as well as precludes H3 Lys-9 methylation by SUV39H1 (31, 51). In conclusion, the results presented in this paper further extend the histone code hypothesis and suggest that Set1-mediated H3 Lys-4 methylation primarily acts in the maintenance of active chromatin configurations at euchromatic chromosomal domains in fission yeast.

Acknowledgments

We thank John Aris for providing Nop1 antibody, and Keith Gull for TAT1 antibody. We also thank Rui-Ming Xu, Ira Hall, Asra Malikzay, and Gurumurthy Shankaranarayana for critical reading of the manuscript, and Winship Herr for helpful discussions. This work was supported by National Institutes of Health Research Grant GM59772.

Abbreviations

SET

Su(var)3–9 enhancer-of-zeste and trithorax

ChIP

chromatin immunoprecipitation

RRM

RNA recognition motif

YEA

yeast extract adenine

FOA

5′-fluoroorotic acid

DAPI

4′,6-diamidino-2-phenylindole

Footnotes

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “Self-Perpetuating Structural States in Biology, Disease, and Genetics,” held March 22–24, 2002, at the National Academy of Sciences in Washington, DC.

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