Research Article

Establishment and Maintenance of a Heterochromatin Domain

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Science  27 Sep 2002:
Vol. 297, Issue 5590, pp. 2232-2237
DOI: 10.1126/science.1076466

Abstract

The higher-order assembly of chromatin imposes structural organization on the genetic information of eukaryotes and is thought to be largely determined by posttranslational modification of histone tails. Here, we study a 20-kilobase silent domain at the mating-type region of fission yeast as a model for heterochromatin formation. We find that, although histone H3 methylated at lysine 9 (H3 Lys9) directly recruits heterochromatin protein Swi6/HP1, the critical determinant for H3 Lys9 methylation to spread in cis and to be inherited through mitosis and meiosis is Swi6 itself. We demonstrate that a centromere-homologous repeat (cenH) present at the silent mating-type region is sufficient for heterochromatin formation at an ectopic site, and that its repressive capacity is mediated by components of the RNA interference (RNAi) machinery. Moreover, cenH and the RNAi machinery cooperate to nucleate heterochromatin assembly at the endogenous matlocus but are dispensable for its subsequent inheritance. This work defines sequential requirements for the initiation and propagation of regional heterochromatic domains.

In eukaryotes, the organization of chromatin into higher-order structures governs diverse chromosomal processes. Besides creating distinct metastable transcriptional domains during cellular differentiation, the formation of higher-order chromatin domains is widely recognized to be essential for imprinting, dosage compensation, recombination, and chromosome condensation (1–4). The assembly of heterochromatin at centromeres is essential for the accurate segregation of chromosomes during cell division, and the formation of such specialized structures at telomeres protects chromosomes from degradation and from aberrant chromosomal fusions (2). Moreover, repetitive DNA sequences such as transposable elements are often assembled into heterochromatin that, in addition to its role in transcriptional repression, maintains genome integrity by suppressing recombination between repetitive elements (5).

The posttranslational modification of histone tails plays a causal role in the assembly of higher-order chromatin, and accumulating evidence suggests that patterns of histone modification specify discrete downstream regulatory events (6, 7). The factors that define particular chromosomal domains as preferred sites of heterochromatin assembly are largely uncharacterized. It has been suggested that heterochromatin protein complexes are preferentially targeted to repetitive DNA elements, such as commonly found at the pericentric heterochromatin and intergenic regions of higher eukaryotes (8, 9). Interestingly, rather than any specific sequence motif, the repetitive nature of transgene arrays alone appears to be sufficient for heterochromatin formation (9, 10). Furthermore, studies of position effect variegation have shown that heterochromatin complexes possess the ability to spread along the chromosomes, resulting in the heritable inactivation of nearby sequences (2).

Higher-order chromatin structure is critical for the functional organization of centromeres and the mating-type region of the fission yeast Schizosaccharomyces pombe (2). At centromeres, tandem and inverted arrays of the dg anddh centromeric repeats surrounding the unique central core are assembled into heterochromatin and are bound by CENP-B proteins that resemble the transposase encoded by POGO-like elements (11–14). At the mating-type region, a 20-kb domain containing the mat2 and mat3 silent donor loci and the interval between them, known as theK-region, are subject to heterochromatin-mediated silencing and recombinational suppression (2). Heterochromatin formation at the centromeres and within the silent mating-type (mat2/3) interval requires many of the same trans-acting factors, including histone deacetylases (HDACs); the H3 Lys9–specific methyltransferase Clr4; and Swi6, the fission yeast counterpart to mammalian HP1 (13, 1518).

We previously showed that formation of heterochromatin within the entire 20 kb of the silent mating-type region depends on H3 Lys9 methylation by Clr4 and the subsequent binding of Swi6, both of which are restricted to this domain by the IR-R and IR-L boundary elements (19). The K-region, in particular the 4.3-kb cenH sequence that contains several clusters of short direct repeats and shares strong homology withdg and dh centromeric elements, is important for heterochromatin assembly (20). Replacement of thecenH-containing region with ura4 +(KΔ::ura4+ ) results in a metastable locus that displays alternative silenced (ura4-off) and expressed (ura4-on) epigenetic states (21, 22). This variegation ofura4+ expression is due to defects in establishment of the silenced chromatin state inKΔ::ura4+ cells. Once assembled, the ura4-off state is remarkably stable during both mitosis and meiosis. Moreover, maintenance of theura4-off state requires functional Swi6, Clr4, and HDACs.

Maintenance of H3 Lys9 methylation depends on Swi6.

Our previous findings predicted a model for heterochromatin formation in which the cooperative activity of HDACs and the H3 Lys9methyltransferase Clr4 establish a “histone code” that is essential for localization of Swi6 to silenced genomic locations (18). Although H3 Lys9 methylation is required for the chromatin association of Swi6, mutations in Swi6 had minimal effects on levels of H3 Lys9 methylation at theKint2::ura4+ reporter gene located within the cenH repeat, indicating that H3 Lys9methylation acts upstream of Swi6 (18) (Fig. 1A). However, we discovered that, inKΔ::ura4+ cells lacking the cenH repeat, the presence of H3 Lys9methylation strictly depends on Swi6, as shown by chromatin immunoprecipitation (ChIP) experiments (Fig. 1B). This indicates that a portion of the sequence deleted in theKΔ::ura4+ strain has the ability to recruit H3 Lys9 methylation by itself and that flanking sequences present in theKΔ::ura4+ strain are capable of recruiting and maintaining H3 Lys9 methylation only in the presence of Swi6.

Figure 1

Swi6 is required for H3 Lys9methylation at the mat region. Effects of mutation inswi6 (swi6-115) on Swi6 and H3 Lys9methylation levels at Kint2::ura4+ (A) andKΔ::ura4+ (B). Schematic representations ofKint2::ura4+ withura4+ inserted at the cenH region andKΔ::ura4+ carrying replacement of the cenH-containing region withura4+ are shown (top). Levels of H3 Lys9 methylation and Swi6 were determined by ChIP as described (21). DNA isolated from immunoprecipitated chromatin fractions with antibodies against Swi6 and H3 Lys9-methyl (K9) or from whole-cell extract (WCE) was quantitatively analyzed with a competitive polymerase chain reaction (PCR) strategy, in which one primer-pair amplifies different-sized PCR products from the full-lengthura4+ at Kint2::ura4 + orKΔ::ura4+ locations and the control ura4DS/E minigene at the endogenous location. PCR fragments were resolved on polyacrylamide gels and quantified with a PhosphorImager. The ratios ofura4+ and ura4DS/E signals in the ChIP and WCE lanes were used to calculate the relative precipitated fold enrichment, shown below each lane. (C) Swi6 and H3 Lys9 methylation (K9) are differentially localized in theura4-off and ura4-on epialleles. ChIP was performed on KΔ::ura4+ cells differing only in the epigenetic state of their matlocus. (D) Multiple copies of swi6+ cause an increase in H3 Lys9 methylation levels atKΔ::ura4+. Three copies of swi6+ (swi6+-333) or six copies of clr4+ (clr4+-666) inserted at their endogenous chromosomal location were combined with the ura4-onepiallele through genetic crosses. The quantitative measurement of Swi6 and H3 Lys9 methylation (K9) levels was carried out with ChIP.

The KΔ::ura4+ ura4-offcells exhibit considerably higher levels of Swi6 throughout theirmat2/3 region than ura4-on cells (21). Consistent with these findings, we observed higher levels of H3 Lys9 methylation at the mating-type region inura4-off cells (Fig. 1C). Additional copies ofswi6+ resulted in increases in H3 Lys9 methylation and Swi6 levels at the mat2/3region, further supporting the possibility that Swi6 promotes H3 Lys9 methylation at the mat locus (Fig. 1D). The increase in levels of Swi6 and H3 Lys9 methylation also correlates with an increase in ura4-on toura4-off conversion. Interestingly, multiple copies ofclr4+ do not cause considerable changes in H3 Lys9 methylation and Swi6 localization. These results underscore the importance of Swi6 in the maintenance of H3 Lys9 methylation and heterochromatin in the absence of thecenH repeat and are consistent with our previous work suggesting that Swi6 protein is a critical component of the epigenetic imprint (21).

The interdependence of Swi6 and H3 Lys9 methylation at the mat2/3 region suggests an “epigenetic loop” for inheritance of the heterochromatic state, whereby H3 Lys9methylation and Swi6 mutually support their own maintenance in a self-perpetuating manner. This mechanism would predict that differential localization of Swi6 and H3 Lys9 methylation patterns defining ura4-on and ura4-off epialleles would be inherited in cis and maintained even when these epialleles are combined into the same environment of a diploid nucleus. To test this possibility, we crossed ura4-off and ura4-onstrains differing at the his2 marker tightly linked to themat locus (ura4-off his2 ;ura4-on his2+ ). Sporulation and tetrad analysis of the resulting diploid showed a 2 Ura+ His+: 2 Ura His segregation pattern in each tetrad, indicating that the epigenetic state of the matregion is inherited in cis and segregates as a marker linked to the respective his2 alleles (Fig. 2) (22). Consistent with the ability of heterochromatin complexes to maintain themselves, ChIP analysis showed that the different levels of H3 Lys9methylation and Swi6 localization corresponding to theura4-on and ura4-off states are inherited in cis and are stable through meiosis (Fig. 2).

Figure 2

Differential H3 Lys9 methylation and Swi6 localization patterns shown byura4-off and ura4-on epialleles are stably inherited in cis. Diagram of the cross is shown (top).KΔ::ura4+ strains carrying ura4-off (his2 ) andura4-on (his2+ ) epialleles differing at the tightly linked his2 marker were crossed, allowed at least 30 generations of diploid growth, sporulated, and subjected to tetrad analysis. Resulting colonies were replicated onto nonselective medium (N/S), medium lacking uracil (AA-URA), medium lacking histidine (AA-HIS), or medium containing 5-fluoroorotic acid (FOA), which selects for the growth of Ura cells. Segregants from individual tetrads were subjected to ChIP analysis with Swi6 and H3 Lys9-methyl antibodies (bottom).

cenH is a nucleation center.

Genetic studies with theKΔ::ura4+ reporter highlighted the requirement of cenH in efficient establishment of the silenced chromatin state at the matregion (20). Furthermore, the persistence of H3 Lys9 methylation atKint2::ura4+ in swi6 mutant cells (see Fig. 1A) led us to hypothesize that heterochromatin formation is initiated at the cenH repeat but requires Swi6 to spread across the entire silenced domain. This hypothesis predicts that H3 Lys9 methylation will be restricted tocenH in the absence of Swi6. To test this, we performed high-resolution ChIP analysis of H3 Lys9 methylation and H3 Lys4 methylation [a modification associated with active chromatin (19)] at the mat region in wild-type and swi6 mutant strains (Fig. 3). We found that H3 Lys9 methylation in the swi6 mutant strain was restricted to a small portion of the mat region encompassing cenH. The loss of H3 Lys9methylation at the mat2/3 interval was correlated with only a slight increase in H3 Lys4 methylation, although we did observe a small peak directly at the transcribed portion of cenH(Fig. 3) (23). These data indicate that the recruitment of H3 Lys9 methylation to the cenH repeat region occurs via a Swi6-independent mechanism and suggest that Swi6 is required for the spreading and maintenance of H3 Lys9methylation across the rest of the silent mat2/3 interval.

Figure 3

Effects of mutation in swi6 on H3 Lys9 and H3 Lys4 methylation at themat2/3 interval. A physical map of the silent mating-type region is shown (top). High-resolution mapping of H3 Lys9 or H3 Lys4 methylation was carried out as described (19). ChIP with antibodies to methylated H3 Lys9 or H3 Lys4 was used to measure H3 methylation levels at respective sites throughout the mat2/3interval. DNA isolated from ChIP and WCE fractions was subjected to multiplex PCR to amplify DNA fragments from the mat locus as well as an act1 fragment serving as an internal amplification control. The ratios of the mat locus and control act1 signals present in WCE were used to calculate relative fold enrichment of precipitated samples. Quantitation of these results is plotted in alignment with a map of the mat locus. WT, wild type.

The results presented above suggest that cenH directly participates in heterochromatin formation by promoting recruitment of histone-modifying enzymes and Swi6. To test this, we examined the contribution of cenH to heterochromatin formation at an ectopic, otherwise euchromatic site. We used a strain in which the 3.6-kb cenH repeat fused to an ade6+ reporter gene (cenH-ade6+ ) was inserted into theura4 gene. In wild-type cells, cenH confers repression on the reporter gene and results in a variegated expression phenotype (Fig. 4A) (24). Silencing at the ectopic site depends on functional Clr4 and Swi6 and correlates with preferential enrichment of both H3 Lys9 methylation and Swi6 (Fig. 4B). Similar to themat region, H3 Lys9 methylation at the ectopiccenH-ade6+ location occurs independently of Swi6. These data demonstrate that the cenH repeat is sufficient to induce heterochromatin formation and that it requires similar chromatin modification and trans-acting factors as the endogenous mat region.

Figure 4

cenH-mediated heterochromatin formation at an ectopic site requires RNAi machinery. (A) Deletions of the components of RNAi machinery suppress variegation ofcenH-ade6+ expression. Schematic representation of the cenH-ade6+ construct inserted into theura4 locus is shown (top). Strains carryingcenH-ade6+ in ago1Δ,dcr1Δ, or rdp1Δ deletion background were constructed by genetic crosses. The cenH-ade6+ expression phenotypes were scored by a colony color assay. Cells were plated on adenine-limiting yeast extract medium and incubated at 33°C for 3 days before photography. The red or white color of colonies implies Ade or Ade+ phenotype, respectively. Wild-type cells display 21% red, 11% pink, and 68% white colonies, whereas mutants exhibited only white colonies. (B) ChIP analysis of the ectopiccenH-ade6+ with Swi6 and H3 Lys9-methyl antibodies in wild-type (WT) and RNAi deletion strains. The primer pair amplifies different-sized fragments from the cenH-ade6+ locus and theade6DN/N minigene at the endogenous location.

cenH-mediated silencing requires the RNAi machinery.

RNA interference (RNAi) is a mechanism through which double-stranded RNA (dsRNA) silences cognate genes (25). The dsRNA serves as a sequence-specific trigger for destruction of homologous RNAs and has been shown in some cases to result in the epigenetic silencing of homologous genes (26, 27). S. pombecontains homologs of the Argonaute (ago1), Dicer (dcr1), and RNA-dependent RNA polymerase (rdp1) genes required for RNAi-related processes in other systems (25,28, 29). Our recent analysis showed silencing and heterochromatin assembly at centromeric repeats in fission yeast require these factors (30). Interestingly, we found that deletions of ago1, dcr1, or rdp1 abolished repression of thecenH-ade6+ reporter (Fig. 4A). Furthermore, ChIP analysis revealed that the locus was no longer able to recruit and/or maintain H3 Lys9 methylation and Swi6 (Fig. 4B). These data indicate that cenH-induced silencing and the corresponding H3 Lys9 methylation and Swi6 localization to the ectopic domain require the RNAi machinery.

RNAi machinery is required for initiation of heterochromatin.

To investigate the role of the RNAi machinery in heterochromatin assembly at the endogenous mat region, we introduced theKint2::ura4+ reporter into the respective mutant backgrounds with genetic crosses. Surprisingly, silencing at the mat locus was intact in the mutant strains (Fig. 5A), and the efficiency of mating-type interconversion, which depends on the heterochromatic structure at the mat2/3 region, was unaffected. More importantly, the levels of Swi6 protein and H3 Lys9methylation at the mat2/3region were comparable in wild-type and mutant strains. These analyses suggest that the RNAi machinery is dispensable for maintaining a preassembled heterochromatic state.

Figure 5

The RNAi machinery is required for initiation of heterochromatin at the mating-type locus. (A) RNAi mutants are dispensable for the maintenance of heterochromatin atKint2::ura4 +. A heterochromaticKint2::ura4+ was introduced into theago1Δ, dcr1Δ, and rdp1Δ mutant backgrounds by genetic crosses. Serial dilution plating assays (top) in the presence and absence of FOA were performed to measureKint2::ura4+ expression. Levels of Swi6 and H3 Lys9 methylation atKint2::ura4+ in the strains were determined by ChIP analysis. (B) RNAi mutants are defective in the establishment of heterochromatin. Wild-type and mutant strains were treated with 35 μg of TSA per ml for 10 generations and were allowed to grow for an additional 10 generations in the absence of TSA. Expression of Kint2::ura4+ is shown by serial dilution analysis (top). Levels of Swi6 and H3 Lys9 methylation atKint2::ura4+ after recovery from TSA treatment were determined by ChIP. (C) RNAi mutants cannot efficiently initiate heterochromatin formation. A mating-type region derived from wild-type or clr4Δ background was introduced into the RNAi mutant backgrounds by genetic crosses (top). Diploids were constructed by crossing the indicated strains, sporulated, and subjected to tetrad analysis. ThernaiΔclr4+Kint2::ura4+ segregants from each cross were assayed for silencing and efficiency of mating-type switching, which also depends on heterochromatin assembly at the mat locus. To assay mating-type switching, colonies were replicated onto sporulation medium (PMA+) and stained with iodine vapors. Dark staining indicates efficient mat switching, and light or sectored staining indicates defects in switching and heterochromatin formation. Levels of Swi6 and H3 Lys9 methylation at themat locus of the indicated cultures were assayed by ChIP.

We next addressed the role of RNAi components in the establishment of silencing by examining their involvement in the initiation step of heterochromatin formation. The deacetylase inhibitor trichostatin A (TSA) has previously been shown to erase the epigenetic imprint governing silencing at the mat region (17). We observed that treatment of wild-type and mutant cells with TSA for 10 generations alleviatedKint2::ura4+ silencing in most cells. After an additional 10 generations of growth in the absence of TSA, wild-type cells fully reestablished silencing at the mat2/3locus (Fig. 5B). In striking contrast, ago1Δ,dcr1Δ, and rdp1Δ strains were defective in the establishment of heterochromatin at the mat2/3 locus because only a relatively small proportion of cells acquired silencing of Kint2::ura4+ (Fig. 5B).ChIP analysis demonstrated that, after recovery from TSA treatment, the levels of H3 Lys9 methylation and Swi6 were considerably higher at the mat region of wild-type cells compared with RNAi mutant cells (Fig. 5B). This indicates that the RNAi deletion strains are unable to efficiently establish the heterochromatic state after it has been erased.

To genetically test the role of RNAi machinery in the initiation of heterochromatin formation, we used a clr4Δ strain carrying the Kint2::ura4+ reporter gene to construct diploid strains heterozygous for clr4Δ and homozygous for ago1Δ, dcr1Δ, orrdp1Δ (for example, clr4+/clr4Δdcr1Δ/dcr1Δ). Because Clr4 is the sole H3 Lys9–specific methyltransferase in fission yeast, the mat locus propagated in a clr4Δ background is completely devoid of H3 Lys9 methylation and Swi6 protein (18). Diploids were sporulated to obtainago1Δ clr4+ ,dcr1Δ clr4+ , orrdp1Δ clr4+ haploid segregants. Phenotypic analysis of the segregants that contain a functional H3 Lys9 methyltransferase but lack RNAi machinery revealed severe defects in heterochromatin assembly at the mat locus, as observed by both Kint2::ura4+ expression and the decreased efficiency of mating-type interconversion (Fig. 5C). ChIP analysis showed that the RNAi mutant strains containing a mat region derived from the clr4Δ background have less H3 Lys9 methylation and Swi6 protein than strains in which the mat region was derived from a wild-type background (Fig. 5C). It is noteworthy, however, that after introduction of the clr4+ allele the proportion of cells with a silenced mat region increases each generation in an inefficient and highly stochastic manner, indicating that heterochromatin formation eventually does occur in the absence of the RNAi machinery, likely via an alternative Swi6-based mechanism.

These data indicate that, although the RNAi mutant strains are able to maintain a silenced mat region when it is derived from a wild-type parent, they are unable to effectively establish silencing at a mat region rendered epigenetically active by TSA treatment or propagation in a clr4Δ background. These findings are consistent with the inefficient establishment of silencing observed inKΔ::ura4+ cells carrying deletion of cenH (20) and further suggest that the RNAi machinery and cenH repeat operate in the same pathway to establish heterochromatin.

A model for heterochromatin assembly.

The results presented here define sequential events in the assembly of heterochromatin at the mating-type region of fission yeast. We show that the establishment of epigenetic silencing requires an initial nucleation event and is distinct in this respect from mechanisms that act in cis to reinforce propagation of the heterochromatic state. Supporting our identification of cenHas an RNAi-dependent heterochromatin nucleation center, a portion ofcenH homologous to the dh repeat is the minimal sequence sufficient for silencing at an ectopic site (24). This sequence is located within the transcribed region of cenH (23) and is homologous to some of the recently described heterochromatic siRNAs (31). In our current model, transcripts derived fromcenH are processed by the RNAi machinery, and resulting RNA intermediates directly recruit HDAC and H3 Lys9methyltransferase activities to the mat locus. This may occur through an interaction between siRNA and the chromodomain (32) of Clr4 and/or the WD-40 repeat containing protein Rik1, also required for H3 Lys9 methylation (18). This initial recruitment is proposed to nucleate heterochromatin by creating H3 Lys9 methylated binding sites for Swi6 (33). Once bound to chromatin, Swi6 serves as a platform for the recruitment of histone-modifying activities that create additional Swi6 binding sites on adjacent nucleosomes, thus enabling spreading to occur in a stepwise manner (2, 21). The presence of chromatin boundaries ensures that heterochromatin does not spread to neighboring euchromatic regions (19). Upon chromosome replication, parental histone H3 (34) and Swi6 are hypothesized to segregate randomly to the daughter chromatids, and Swi6-based activities serve to imprint the parental histone modification pattern onto newly assembled nucleosomes by the same mechanism with which they promote spreading in cis. Because heterochromatin formation eventually occurs without the RNAi machinery, and because stochastic initiation events also occur in the absence ofcenH, we predict the existence of additional Swi6-dependent, RNAi-independent nucleation sites in the mat region.

The mechanism proposed above is reminiscent of mammalian X-chromosome inactivation, in which an H3 Lys9 methylation hotspot upstream of the Xist locus serves to initiate the cooperative spreading of Xist noncoding RNA and H3 Lys9 methylation across the entire inactive X (35). Significantly, once silencing is established, Xist RNA becomes dispensable and the heterochromatic state persists in the absence of the initial stimulus (3). Mechanisms involving RNAi-like processes may also operate in the lineage-specific establishment of silenced chromatin domains during development.

It is remarkable that, in fission yeast, the mating-type locus appears to have used a repetitive DNA element to organize a highly specialized chromatin structure that controls transcriptional silencing, recombinational suppression, and the nonrandom utilization of silent cassettes during mating-type switching. Similar processes may influence a variety of chromosomal functions important for preserving genomic integrity, such as prohibition of wasteful transcription and suppression of deleterious recombination between repetitive elements. In this regard, it should be noted that the presence of large, repetitive heterochromatic regions is widespread among eukaryotes, and, in Drosophila, plants, mammals, and some fungi, the introduction of repetitive sequences of diverse origin can stimulate pathways leading to heterochromatin formation (8, 9, 36,37). Future analysis of the connection between RNAi and chromatin assembly will provide insight into the epigenetic organization of eukaryotic genomes.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: grewal{at}cshl.org

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