Telomere Binding Protein Taz1 Establishes Swi6 Heterochromatin Independently of RNAi at Telomeres

Current Biology, Vol. 15, 1808–1819, October 25, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.09.041

Telomere Binding Protein Taz1 Establishes Swi6 Heterochromatin Independently of RNAi at Telomeres Junko Kanoh,1,* Mahito Sadaie,2 Takeshi Urano,3 and Fuyuki Ishikawa1 1 Department of Gene Mechanisms Graduate School of Biostudies Kyoto University Kitashirakawaoiwake-cho, Sakyo-ku Kyoto 606-8502 Japan 2 Laboratory of Chromatin Dynamics RIKEN Center for Developmental Biology 2-2-3 Minatojima-Minamimachi, Chuo-ku Kobe 650-0047 Japan 3 Department of Biochemistry II Nagoya University Graduate School of Medicine 65 Tsurumai, Showa-ku Nagoya 466-8550 Japan

Summary Background: The telomere is a specialized heterochromatin conserved among eukaryotes. However, it remains unknown how heterochromatin protein 1 (HP1) is recruited to telomeres and how telomere heterochromatin is formed. In fission yeast, the RNAi (RNA interference)-RITS (RNA-induced initiation of transcriptional silencing) pathway initiates heterochromatin formation at the centromeres and the silent mat locus by using common DNA sequences, the dg and dh repeats, as the templates for small interfering RNA (siRNA). Results: We found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family. Additionally, Swi6 heterochromatin is established by a part of the subtelomere that contains sequences highly homologous to that of the dh repeat, and it is strikingly destabilized by the deletion of both Taz1 and RNAi-RITS. Transcripts from the telomeric dh-homologous region were specifically associated with RITS, and deletion of the telomeric dh-homologous region showed the phenotype similar to that of the rnai mutant in terms of the telomeric silencing, indicating that the RNAi-RITS pathway acts at the telomeric dh-homologous region to establish Swi6 heterochromatin. Furthermore, we found that Taz1 establishes Swi6 heterochromatin independently of the telomeric repeats and the RNAi-RITS pathway at the subtelomeres. Conclusion: The telomere heterochromatin is regulated by at least two factors: One is Taz1, which is telomere specific, and the other is RNAi-RITS, which is commonly used at the constitutive heterochromatin regions.

*Correspondence: [email protected]

Introduction The telomere is a specialized heterochromatin localized at a chromosome end, and it contributes to the many aspects of genome integrity. In fission yeast, the Taz1 protein binds directly to the telomeric repeats and recruits spRap1 or spRif1 to the telomeres [1–3]. These telomere-specific protein complexes are involved in a number of telomere functions, including heterochromatin maintenance at the chromosome end, telomere length control, and telomere clustering toward the spindle pole body (SPB) at premeiotic prophase [1–4]. The expression of the marker gene inserted between the telomeric repeats and the subtelomere is repressed by the higher order of the chromatin structure [1]. In the taz1 and rap1 mutants, however, the expression is significantly derepressed [1, 3], suggesting that the Taz1spRap1 complex directly regulates chromatin structure at the distal end of the telomeres or that it controls the localization of gene-silencing factors. It has been shown that Swi6, a homolog of fruit fly and mammalian HP1, is localized at the telomeres as well as at the centromeres and the silent mat locus [5, 6]. RNAi is involved in the establishment of Swi6 heterochromatin at the centromeres and the silent mat locus [7, 8]. There exists a tandem array of the dg and dh repeats at the pericentromeric otr region [9]. In the K-region of the silent mat locus, there is a DNA sequence (4.3 kb) called cenH, which is highly homologous to the sequences of the partial dg and dh repeats [10]. dsRNA is produced by Rdp1, an RNA-dependent RNA polymerase, and other RNA polymerases, such as RNA polymerase II, with the dg and dh repeats as templates, and Dcr1 (Dicer) digests the dsRNA fragments into 22 to 25 nucleotides of siRNA [11–15]. Ago1 (Argonaute) associates with the Tas3 and Chp1 proteins and with siRNA to form a RITS complex, which induces the methylation of histone H3 at Lys9 (K9) by Clr4 by targeting siRNA to the homologous sequence, and then Swi6 is recruited to the methylated histone H3-K9 [16, 17]. Recently, it has been shown that the ATF/CREB family’s Atf1/Pcr1 transcription factors, which function downstream of Spc1/Sty1 MAP kinase [18–21], are also involved in the establishment of Swi6 heterochromatin at the silent mat locus [22]. Atf1/Pcr1 recruits Swi6 at the cAMP-response element (CRE) motifs that are located outside cenH in the K-region. The deletion of either Atf1 (or Pcr1) or an RNAi component does not cause a significant defect in the establishment and maintenance of Swi6 heterochromatin at the silent mat locus; however, the deletion of both Atf1 (or Pcr1) and an RNAi component causes a severe defect in the establishment of Swi6 heterochromatin. Thus, RNAi-RITS and Atf1/Pcr1 function in redundant pathways at the silent mat locus. Furthermore, the localization of Swi6 is significantly reduced in the atf1 (or pcr1) rnai double mutant, indicating that the removal of major establishment factors destabilizes Swi6 heterochromatin. Once Swi6 is recruited to a specific region, it selfassembles and spreads into its neighboring regions by interacting with histone-modifying enzymes that create

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Swi6 binding sites on adjacent nucleosomes [16, 23]. Therefore, the factors that are required only for the establishment of Swi6 heterochromatin are dispensable for the spreading of Swi6. The components of RITS are also stably associated with the constitutive heterochromatin regions in a histone H3-K9 methylation (Clr4)dependent manner and promote both transcriptional and posttranscriptional silencing [13, 24–26]. RITS is recruited to cenH at the silent mat locus in Dcr1-dependent and Swi6-independent manners, whereas it spreads throughout the entire silenced mat region in Dcr1-independent and Swi6-dependent manners [13]. In Drosophila, HP1 is required for the many aspects of telomere functions, including telomere-position effect, telomere-length control, and inhibition of telomere fusion [27–29]. The Drosophila telomeres contain arrays of mobile retrotransposon-like elements instead of telomeric repeats at the termini [30]. The fact that HP1 is localized at the telomeres that lack telomeric retrotransposons suggests that HP1 recognizes some aspects of the telomere that are distinct from the DNA sequence of the telomere end [27]. In mouse, heterochromatin is involved in telomere length control. Suv39h1 and Suv39h2 are the methyltransferases specific for histone H3-K9 [31]. In the Suv39h double null mouse, which has a decreased level of histone H3-K9 methylation and reduced binding of HP1 homologs to the chromosome, the telomeres are abnormally elongated [32]. Although studies of fly and mouse have revealed some functions of HP1 homologs at the telomeres, it remains unclear how HP1 homologs are related to telomere-specific proteins and how telomere heterochromatin is formed in eukaryotes. In this study, we uncovered a mechanism of heterochromatin formation at the telomeres in fission yeast. We show that the telomeric repeats are sufficient for the establishment of Swi6 heterochromatin and that Taz1 and Clr4 are required for it. Additionally, the telomeres have another cis element for heterochromatin establishment at the subtelomeres. This element contains cenH-like sequences and is regulated by the RNAi-RITS pathway. Furthermore, Taz1 establishes Swi6 heterochromatin at the subtelomeres independently of telomeric repeats and RNAi-RITS. We propose a model to explain how constitutive heterochromatin is stably inherited. Results Swi6 Is Localized at the Long Region of the Subtelomere To investigate the relationship between the telomerespecific Taz1 complexes and Swi6, we first examined gene silencing at the subtelomere (Figure 1A). The ura4+ marker was inserted at loci that were w7, 11, 33, 52, or 56 kb from the telomeric repeats on the right arm of chromosome II. The ura4+ marker was also inserted at the distal end (0 kb) of the telomere-associated sequence (TAS) on the left arm of chromosome II (see the Supplemental Data for the fission-yeast genome sequence). Growths on SD-uracil and 5-FOA plates were used to monitor ura4+ expression and repression, respectively. In the wild-type, repression of ura4+ expression was detected up to 52 kb from the telomeric re-

peats, indicating that telomere heterochromatin spreads approximately 50 kb from the chromosome end on the right arm of chromosome II. Deletion of taz1+ derepressed the expression of ura4+ at 0 and 7 kb positions. Deletion of rap1+ derepressed the expression of ura4+ only at the distal end (0 kb) of the TAS. In contrast, deletion of swi6+ had effects on the silencing throughout the long region, from the distal end of the TAS to the locus at 52 kb from the telomeric repeats. These results suggested that Taz1 and spRap1 are localized only around the telomere end, whereas Swi6 is localized at the long region of the subtelomere. To investigate the localization of telomere binding proteins at the entire telomere region, we next performed chromatin immunoprecipitation (ChIP) assays (Figure 1B). Because the TASs of chromosomes I and II are highly homologous, the relative enrichment from the distal end of the TAS to the locus at 45 kb from the chromosome end shown in Figure 1B probably applies to all the arms of chromosomes I and II. On the other hand, the relative enrichment from the locus at 45 kb to 50 kb from the chromosome end applies only to the left arm of chromosome I and both arms of chromosome II, and that from the locus at 50 kb to 70 kb from the chromosome end applies only to the right arm of chromosome II. Taz1 and spRap1 were highly enriched at the telomere end. Taz1 was also localized at the distal end of the TAS spanning approximately 10 kb, consistent with our previous study [33]. The localizations of Taz1 and spRap1 correlate with the silencing defects in the taz1 and rap1 mutants shown in Figure 1A. The localization of spRap1 at the telomere was strikingly reduced by the taz1 deletion, which is also consistent with our previous study [3]. In contrast, Swi6 was not significantly enriched at the distal end of the TAS; rather, it was localized at the long region of the subtelomere spanning approximately 45 kb on the right arm of chromosome II and possibly on the left arms of both chromosomes I and II (Figure 1B, data not shown). Swi6 was localized at the subtelomere spanning approximately 75 kb on the right arm of chromosome I (Figure 1C). The localization of Swi6 at the telomeres of chromosome III will be described elsewhere. Note, however, that the telomere localization of Swi6 was dramatically decreased by the clr4 deletion (Figure 1B). These data are consistent with those of the recent DNA microarray analyses [25]. Together, these results indicated that the localization pattern of Swi6 is different from that of Taz1 or spRap1 and that the Swi6 localization at the entire telomere region is dependent on the methylation of histone H3-K9 by Clr4. The telomere localization of Swi6 was not significantly affected by the taz1 deletion (Figure 1B), and the localization of Taz1 was not significantly altered by the swi6 deletion (Figure 1D), indicating that their localizations are mutually independent and that Taz1 is dispensable for the maintenance of pre-existing Swi6 heterochromatin at native telomeres. Telomeric Repeats Are Sufficient for the Establishment of Telomere Heterochromatin To investigate which part of the telomere is required for the establishment of Swi6 heterochromatin, we first determined whether the telomeric repeats could estab-

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Figure 1. Swi6 Is Localized at the Long Region of the Subtelomere (A) Taz1 and Swi6 have different effects on gene silencing at the telomeres. The ura4+ marker was inserted at various loci of the subtelomeres. Numbers on the right indicate the approximate distance from the telomeric repeats. Serial-dilution plating assays were performed to monitor ura4+ expression on YES (complete), SD-uracil (lacking uracil), and 5-FOA (YES with 5-FOA) plates. (B) ChIP analyses of the localizations of Taz1-HA, spRap1-HA, and Swi6 on the right arm of chromosome II. (C) ChIP analysis of the localization of Swi6 on the right arm of chromosome I. (D) ChIP analyses of the localization of Taz1-HA in the wild-type and swi6⌬. Error bars indicate the standard error from multiple independent experiments.

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Figure 2. Telomeric Repeats Are Sufficient for the Establishment of Swi6 Heterochromatin (A) Schematic representation of the DNA fragments inserted at the ade6 locus of the Type B circular chromosome. The fragment of the telomeric repeats is w300 bp long, the TAS is w10 kb long, and the lambda DNA is w11 kb long. In the ade6::TEL-TAS strain, the ura4+ marker is located 0.7 kb from the telomeric repeats, whereas that in the ade6::TEL-lambda DNA strain is located 0.25 kb from the telomeric repeats. The ade6::ura4+ strain was used as control. (B) Swi6 is recruited near the telomeric repeats inserted at the ade6 locus. ChIP analyses of the Swi6 localization at the ura4+ locus and at cenH in the silent mat locus are shown. act1+ was used as internal control. (C) Telomeric repeats are sufficient for the formation of silent heterochromatin. Serial-dilution plating assays were performed on EMM+AA (containing uracil), EMM-uracil (lacking uracil), and 5-FOA (EMM with uracil and 5-FOA) plates. (D and E) Telomeric repeats are sufficient for the formation of a telomere-specific complex functional for telomere clustering in meiosis. The frequency of the cos1322 (ade6+)-Sad1 (SPB) association at the horsetail stage was measured by fluorescence in situ hybridization. Fifty cells at the horsetail stage were analyzed for each strain.

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Figure 3. Taz1 Is Required for the Establishment of Swi6 Heterochromatin near the Telomeric Repeats (A) Swi6 is not recruited near the telomeric repeats inserted at the ade6 locus in taz1⌬. Each gene was deleted, and then TEL-TAS (10 kb) with ura4+ was inserted at the ade6 locus. ChIP analyses of the Swi6 localization at the ura4+ locus and at cenH in the silent mat locus are shown. (B) Swi6 localization near the telomeric repeats inserted at the ade6 locus is destabilized by the taz1 deletion. ChIP analyses of the Swi6 localization at the ura4+ locus and at the dh repeats of the centromeres are shown. Enrichment of K9-methylated histone H3 at the dh repeats of the centromeres is not significantly affected by the chp1 deletion [24]. Error bars indicate the standard error from multiple independent experiments. (C) Taz1 is required for the Swi6 localization at the telomere of the minichromosome. The structure of the minichromosome used in this study is shown at the top. ChIP analyses of the Swi6 localization at the telomere (the ura4+ locus) of the minichromosome and at the dh repeats of the centromeres are shown. Error bars indicate the standard error from multiple independent experiments. (D) Taz1 induces the methylation of histone H3-K9 prior to the recruitment of Swi6. The taz1+, swi6+, or clr4+ genes were deleted before the insertion of TEL-TAS (10 kb) with ura4+ at the ade6 locus. ChIP analyses of the localization of the K9-methylated histone H3 at the ura4+ locus and at the dh repeats of the centromeres are shown. Error bars indicate the standard error from multiple independent experiments. (E) Model of the establishment of Swi6 heterochromatin near the telomeric repeats.

lish Swi6 heterochromatin at the ade6 locus, which is originally a euchromatin region (Figure 2A). ChIP analyses showed that the insertion of a 300 bp fragment of telomeric repeats and the adjacent 10 kb of the TAS or 11 kb of lambda DNA with ura4+ at the ade6 locus of the Type B circular chromosome (see below) led to the efficient recruitment of Swi6, although the insertion of ura4+ alone did not (Figure 2B). The expression of ura4+ was silenced when it was inserted at the ade6 locus with the telomeric repeats, indicating that the newly recruited Swi6 at the ade6 locus could form silent heterochromatin (Figure 2C). The difference between TEL-TAS and TEL-lambda DNA in the Swi6 enrichment and in the silencing of ura4+ may reflect the difference in the location of ura4+ or that in the DNA composition (TAS or lambda DNA). Furthermore, the telomeric repeats inserted at the ade6 locus efficiently clustered toward the SPB at premeiotic prophase, and the clustering frequency was strikingly reduced by the taz1 deletion (Figures 2D and 2E). Because spRap1 that is recruited to telomeres by Taz1 plays a critical role in telomere clustering in meiosis [2, 3], these data suggest that the telomeric repeats inserted at the ade6 locus of the circular chromosome induced the formation of the Taz1spRap1 complex that was functional for telomere clustering toward SPB in meiosis. It is also suggested that

the insertion of telomeric repeats at the ade6 locus induced the Clr4-dependent methylation of histone H3K9 because Clr4 (but not Swi6) is required for the normal telomere clustering in meiosis [34, 35]. Collectively, these results suggested that the insertion of telomeric repeats at the ade6 locus is sufficient for the recruitment of Swi6 as well as Taz1-spRap1 to form the telomere-heterochromatin-like structure in the absence of chromosome end. Taz1 Is Required for the Establishment of Swi6 Heterochromatin near the Telomeric Repeats To investigate which proteins are required for the establishment of Swi6 heterochromatin near the telomeric repeats, at the ade6 locus in the appropriate mutant strains we inserted constructs containing the telomeric repeats and either the adjacent 10 kb of the TAS or 11 kb of lambda DNA (Figure 3A and Figure S1). ChIP analyses showed that Swi6 was not enriched at the ade6 locus in the clr4 mutant. Swi6 enrichment at ade6 was also defective in taz1⌬ cells, whereas there was no severe defect in Swi6 recruitment when rap1+, rif1+, or chp1+ was deleted. These data suggested that Taz1 and Clr4, but not spRap1, spRif1, or RITS, are essential for the establishment of Swi6 heterochromatin near the telomeric repeats. The moderate decrease in Swi6 en-

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Figure 4. Swi6 Heterochromatin Is Maintained by the Distal Region of the TAS Independently of Telomeric Repeats (A) Histone H3-K9 is methylated at the distal region of the TAS in the absence of Swi6. ChIP analyses of the localization of the K9methylated histone H3 on the right arm of chromosome II are shown. The region where histone H3-K9 was significantly methylated in the swi6 cells is shaded in gray. (B) Swi6 is enriched at the TAS of Type A but not Type B. Approximately nine kilobases of telomere DNA is missing in Type A, and w14 kb is missing in Type B. The white arrow indicates the ORF of the telomeric-DNA helicase gene (SPBCPT2R1.08c). Gray boxes in the ORF indicate the two fragments homologous to cenH. Note that the TEL-TAS DNA fragment used for Figures 2 and 3 does not contain the ORF. ChIP analyses of the Swi6 localization at the TAS (16, 20, and 30 kb from the original chromosome end) and at the dh repeats of the centromeres are shown. Error bars indicate the standard error from multiple independent experiments.

richment at ade6 in rap1⌬ and rif1⌬ cells suggested the possibility that spRap1 and spRif1 redundantly contribute to the formation of Swi6 heterochromatin. When taz1+ was deleted in a strain that contained an insertion of the telomeric repeats and 10 kb of the TAS at the ade6 locus, Swi6 was no longer enriched at the ade6 locus (Figure 3B). On the other hand, Swi6 enrichment at the dh repeats of the centromeres was maintained in these cells (Figure 3B). These data indicated that the taz1 deletion destabilized pre-existing Swi6 heterochromatin near the telomeric repeats (Figure 3B). To examine whether Taz1 is also indispensable for the Swi6 localization at the chromosome ends, we constructed a minichromosome that contained no TAS by homologous recombination (Figure 3C). Swi6 was enriched at the telomere of the minichromosome, indicating that the telomeric repeats are sufficient and TAS is dispensable for the establishment of Swi6 heterochromatin at the telomeres. The localization of Swi6 was strikingly reduced at the telomere of the minichromosome when taz1+ was deleted, although Swi6 enrich-

ment at the centromeres was unaffected (Figure 3C). At the silent mat locus, the removal of the major establishment factors RNAi and Atf1/Pcr1 results in the marked destabilization of Swi6 heterochromatin [22], presumably because the Swi6-localization loss caused by repeated DNA replication is faster than the rate of selfreassembly of Swi6 heterochromatin in the absence of its establishment factors. Therefore, these data suggested that Swi6 recruitment by Taz1 is required to prevent the destabilization of Swi6 heterochromatin after repeated DNA replication and cell division. We next examined whether Taz1 directly recruits Swi6 to the telomeres. The taz1+, swi6+, or clr4+ genes were deleted, and then the telomeric repeats and the adjacent 10 kb of TAS were inserted at the ade6 locus (Figure 3D). In the taz1 mutant, the methylation of histone H3-K9 at the ade6 locus was strikingly decreased to the level similar to that in the clr4 mutant, whereas it was only moderately decreased in the swi6 mutant, indicating that Swi6 is dispensable for the Taz1-induced Clr4-dependent methylation of histone H3-K9 at the

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Figure 5. RNAi-RITS Is Involved in the Establishment of Swi6 Heterochromatin at the CenH-Homologous Region of the Telomeres (A) The dcr1 and chp1 mutants are not defective in gene silencing at native telomeres. Silencing of ura4+ at w11 and w33 kb from the chromosome end was examined. Serial cell dilutions of the wild-type, swi6⌬, dcr1⌬, and chp1⌬ were spotted on YES (complete) and 5-FOA (YES with 5-FOA) plates. (B and C) Simultaneous deletion of Taz1 and Dcr1 (or Chp1) does not cause a significant decrease in the Swi6 localization at native telomeres.

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ade6 locus. Figures 1B and 3A indicated that Clr4 plays a critical role in the Swi6 localization near the telomeric repeats as well as at the other subtelomeric region. Collectively, these data suggested that Taz1 does not directly recruit Swi6 at the first step of the establishment of Swi6 heterochromatin; rather, Taz1 induces the Clr4dependent methylation of histone H3-K9 to create a Swi6 binding site (Figure 3E). Swi6 Heterochromatin Is Maintained by the Distal Region of the TAS Independently of Telomeric Repeats Because Figure 1B showed that the localization of Swi6 at the native telomeres was not destabilized by taz1 deletion, we next examined whether telomere regions other than the telomeric repeats are responsible for the establishment of Swi6 heterochromatin. When Swi6 heterochromatin is established at the silent mat locus, Clr4 is recruited to the establishment regions by the RITS complex and Atf1/Pcr1 to methylate histone H3K9 before Swi6 is recruited [13, 24]. In the swi6 mutant cells, histone H3-K9 methylation does not spread outward from the initial establishment region [7]. Thus, the region where histone H3-K9 is methylated in the absence of Swi6 is considered to be a potential initial establishment region. To investigate the initial establishment region, we analyzed the methylation of histone H3-K9 at the subtelomere (Figure 4A). Histone H3-K9 was methylated at the subtelomere spanning w45 kb except for the distal end of the TAS on the right arm of chromosome II, the pattern of which was similar to that of the Swi6 localization. In the swi6 mutant, the methylation of histone H3-K9 was detected only at the 6.5–15 kb region from the chromosome end (shaded in gray). These data are consistent with those of the recent DNA microarray analyses [25]. These results suggested that the subtelomeric 6.5–15 kb region is one of the initial establishment regions of Swi6 heterochromatin. We next examined whether the subtelomeric 6.5–15 kb region is actually involved in the establishment of Swi6 heterochromatin. For this purpose, we used the cells with circular chromosomes, in which telomeric repeats-independent phenomena can be observed. Previously, we have identified two types of circular chromosomes by deleting Trt1 [33]. One is Type A, which lacks approximately 9 kb of telomeric DNA from the chromosome end. The other is Type B, which lacks approximately 14 kb of telomeric DNA (Figure 4B). The telomeres of Type A contain the Taz1 binding region, whereas those of Type B do not [33]. We found that Swi6 was enriched at all the loci of the TAS we exam-

ined in Type A, that is, at 16, 20, and 30 kb from the original chromosome end. In contrast, Swi6 enrichment at the telomeres was dramatically reduced in Type B, although it was normal at the centromeres (Figure 4B). These data suggested that the TAS in the region from 9 to 14 kb from the original chromosome end is able to establish Swi6 heterochromatin. The chromosome of Type B does not exhibit such an activity. It is also suggested that the Swi6 heterochromatin in the region located w14 kb from the chromosome end to the centromere-proximal region is formed by its self-assembly and spreading. There exists an open reading frame (ORF) of a putative DNA helicase (SPBCPT2R1.08c) near the end of the telomeres of Type A, as reported previously [36] (Figure 4B, white arrow). According to the Sanger Centre database, the 3# end of the ORF is located at w10.2 kb from the original chromosome end. Thus, the DNA fragment of the TAS used in Figures 2 and 3 does not contain this ORF. Fission yeast has at least four copies of this ORF on the arms of chromosomes I and II [36]. This ORF contains two fragments (w200 and w300 bp) of DNA sequence at w14 kb from the original chromosome end, and these are highly homologous (91% and 84% identities, respectively) to that of cenH at the silent mat locus rather than to the centromeric repeats (Figure 4B, gray boxes). The cenH region contains the partial dg and dh repeats and the 264 bp and 54 bp cenH-specific insertions [10], and the ORF of the telomeric-DNA helicase contains sequences that are homologous to parts of the dh repeat and a part of the 264 bp insertion. These facts suggested the possibility that the telomeric cenH-homologous DNA is one of the candidates for the cis-acting DNA that establishes Swi6 heterochromatin. RNAi-RITS Pathway Is Involved in the Establishment of Swi6 Heterochromatin at the Telomeric cenH-Homologous Region The dh repeat is the DNA template for the siRNAs at the centromeres and the silent mat locus [8, 11, 13, 25]. A recent study has shown that siRNAs are also produced from the distal region of the subtelomere [25]. Therefore, we examined whether the RNAi-RITS pathway is involved in the establishment of Swi6 heterochromatin at the subtelomeres. Deletion of dcr1 or chp1 did not decrease ura4+ silencing at sites located w11 and w33 kb from the chromosome end (Figure 5A), suggesting that a substantial amount of Swi6 is likely to be localized at the native telomeres in these mutants. We determined whether the deletion of both

ChIP analyses of Swi6 localization at the TAS (w11kb from the chromosome end) and at the dh repeats of the centromeres are shown. Error bars indicate the standard error from multiple independent experiments. (D and E) Simultaneous deletion of Taz1 and Dcr1 (or Chp1) dramatically reduces the Swi6 localization at the TAS of Type A. ChIP analyses of Swi6 localization at the TAS (w14 kb from the original chromosome end) and at the dh repeats of the centromeres are shown. Error bars indicate the standard error from multiple independent experiments. (F) Transcripts of the telomeric DNA helicase gene are enriched in the Chp1-13Myc immunoprecipitates. The chp1-13myc and nontagged strains were fixed and immunoprecipitated by anti-Myc antibodies. Precipitated RNAs were subjected to reverse-transcription reaction and were quantified by PCR with primers that recognize the region indicated by the small white box above the ORF. The primers for lys1+ were used as the quantitative control. Error bars indicate the standard error from multiple independent experiments. (G) Deletion of the telomeric cenH-like sequence has a similar effect to that of dcr1 deletion. ura4+ was inserted at various loci of Type A chromosome shown at the top, and its silencing was assayed.

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Taz1 and RNAi-RITS causes a severe defect in Swi6 localization at the telomeres. Deletion of Taz1 did not cause a significant defect in the Swi6 localization (Figures 1B, 5B, and 5C), indicating that Taz1 is not the sole factor for the establishment of Swi6 heterochromatin at the native telomeres. In contrast, dcr1 and chp1 deletions increased Swi6 enrichment, suggesting that the RNAi-RITS pathway is dispensable for the maintenance of Swi6 heterochromatin at the telomeres and that RITS and Swi6 compete for the K9-methylated histone H3 at the telomeres. The deletion of both Taz1 and Dcr1 (or Chp1) did not cause a significant decrease in Swi6 localization at the native telomeres, but there was a striking destabilization of Swi6 localization at the subtelomeres of Type A (Figures 5B–5E). In the taz1dcr1 or taz1chp1 cells, Swi6 was normally enriched at the centromeres. Thus, the effect of the Taz1 and Dcr1 (or Chp1) deletion on the enrichment of Swi6 is specific for the telomeres. These data indicated that Taz1 and RNAi-RITS establish Swi6 heterochromatin at the telomeres in redundant pathways and that they are the only or at least the major factors for the establishment of Swi6 heterochromatin at the subtelomeres of Type A. It is also suggested that the w9 kb distal region of TAS, a region that is missing in the Type A chromosome, plays an important role in maintaining Swi6 localization independently of Taz1 and RNAi-RITS when it is located at the native telomeres. The recognition of nascent transcripts by siRNAassociated RITS mediates the initial localization of RITS to the specific chromosome regions [12]. We therefore explored whether the RNAi-RITS pathway is directly involved in the establishment of Swi6 heterochromatin at the telomeres. Specifically, we examined whether Chp1 is associated with the transcripts of the telomeric-DNA helicase gene. In comparison to the lys1+ RNA control, the RNAs derived from the forward strand of the telomeric DNA helicase gene were significantly enriched in the Chp1-13Myc immunoprecipitates (Figure 5F). No amplification was detected in the absence of reverse transcriptase (data not shown). These data show that the RITS complex is associated specifically with the transcripts of the telomeric DNA helicase gene. Therefore, it is likely that the RITS complex directly acts at the locus of the telomeric DNA helicase gene. We next examined whether the telomeric cenHhomologous region plays an important role in the establishment of Swi6 heterochromatin. The ura4+ marker was inserted at various loci at the subtelomere of Type A, and its silencing was detected on 5-FOA plates (Figure 5G). When ura4+ was inserted at w33 kb from the original chromosome end (strain e), normal silencing was observed in the wild-type and dcr1 mutant cells, whereas weak silencing was observed in the taz1 mutant. Much weaker silencing was observed in the taz1dcr1 mutant. These data correlated with those of the Swi6 localization shown in Figure 5D. When the telomeric cenH-homologous region was replaced with ura4+ (strain c), silencing was largely absent in the taz1 mutant, indicating that the deletion of the telomeric cenH-homologous region and the dcr1 deletion are equivalent in terms of the subtelomeric silencing. These data also indicate that the RNAi-RITS pathway utilizes the telomeric cenH-homologous region for the estab-

Figure 6. Taz1 Establishes Swi6 Heterochromatin at TAS Independently of RNAi-RITS (A) Scheme of the experiments in (B) and (C). The taz1+ and dcr1+ (or chp1+) genes were deleted simultaneously to remove Swi6 from the TAS of Type A, and then Taz1-HA was reintroduced. (B) ChIP analyses of the localization of Taz1-HA at w9 kb from the original chromosome end. Error bars indicate the standard error from multiple independent experiments. (C) ChIP analyses of the localization of Swi6 at w11 kb from the original chromosome end. Error bars indicate the standard error from multiple independent experiments.

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Figure 7. Model of the Establishment of Heterochromatin in Fission Yeast (A) Establishment and spreading of Swi6 heterochromatin at telomeres. Taz1 and RNAiRITS recruit Swi6 to the distal region of the TAS (the white arrow indicates the ORF of the telomeric DNA helicase). Other unknown factors are possibly involved in the establishment of Swi6 heterochromatin at native telomeres. Swi6 spreads into the centromere-proximal region to cover the subtelomere spanning 45–75 kb. For details, see main text. (B) RNAi-RITS is the common factor for the establishment of Swi6 heterochromatin. Additionally, Taz1 at the telomeres and the CREB family proteins (Atf1/Pcr1) at the mat locus recruit Swi6 to the chromosome. Additional factors specific for Swi6 recruitment at the centromeres are not known. These locus-specific backup systems will stabilize the Swi6 heterochromatin.

lishment of Swi6 heterochromatin. We then examined whether loci other than the cenH-homologous region were important for the Swi6 heterochromatin formation. The ura4+ marker was inserted at w11, w13.3, or w19.3 kb from the original chromosome end (strains a, b, and d). The w19.3 kb locus is outside the transcribed region of the telomeric DNA helicase gene [36]. When taz1+ was deleted, silencing was hardly observed in strains a and b, whereas weak but substantial silencing was observed in the taz1-deleted strain d. These data suggested that the existence of the telomeric cenHhomologous DNA is insufficient and that normal transcription of the telomeric DNA helicase gene is required for the establishment of Swi6 heterochromatin by the RNAi-RITS pathway.

tin at a euchromatin region or at the end of a minichromosome. In both cases, Taz1 plays a critical role in the establishment of Swi6 heterochromatin. Additionally, the fission yeast TAS has another cis element for the establishment of Swi6 heterochromatin. The RNAi-RITS pathway is involved in the establishment of Swi6 heterochromatin at the telomeres by acting at the ORF of the telomeric-DNA helicase containing cenH-homologous sequences. Other unknown factors seem to be involved in the establishment of Swi6 heterochromatin at the native telomeres. After Swi6 is recruited by Taz1 and RNAi-RITS, it spreads into the centromere-proximal region of the subtelomere spanning 45–75 kb (Figure 7A). Discussion

Taz1 Establishes Swi6 Heterochromatin Independently of RNAi-RITS and Telomeric Repeats We next examined whether Taz1 indeed has the ability to establish Swi6 heterochromatin independently of the RNAi-RITS pathway and the telomeric repeats. Taz1 and Dcr1 (or Chp1) were deleted to remove Swi6 from the subtelomeres of Type A, and then Taz1-HA was reintroduced (Figure 6A). Taz1-HA was efficiently recruited to the subtelomeres after the reintroduction, indicating that Taz1 does not require RNAi-RITS, Swi6, or the telomeric repeats for its recruitment to the subtelomeres (Figure 6B). Swi6 was also recruited to the subtelomeres after the reintroduction of Taz1-HA, indicating that Taz1 establishes Swi6 heterochromatin independently of RNAi-RITS and the telomeric repeats (Figure 6C). Conclusions Our analyses indicated that Taz1 and RNAi-RITS establish Swi6 heterochromatin in redundant pathways at the fission-yeast telomeres. Taz1 is localized at the telomeric repeats and the distal end of the TAS, and it induces the methylation of histone H3-K9 by Clr4 to recruit Swi6. The 300 bp fragment of telomeric repeats is sufficient for the establishment of Swi6 heterochroma-

This study demonstrates that a telomere binding protein of the TRF family recruits HP1 to telomeres. Our analyses suggest the possibility that the TRF family proteins in higher eukaryotes are also involved in the establishment of telomere heterochromatin. Taz1 recruits at least three effectors, spRap1, spRif1, and Swi6, to the telomeres; however, the mechanism of the recruitment of Swi6 is different from that of spRap1 or spRif1. Taz1 directly interacts with spRap1 or spRif1 to recruit them to the telomeres [2, 3]. On the other hand, Taz1 induces the methylation of histone H3-K9 by Clr4 to create a Swi6 binding site (Figures 3D and 3E). It has been shown that factors for the establishment of Swi6 heterochromatin other than Taz1, such as RITS and Atf1/Pcr1, also induce the methylation of histone H3K9 by Clr4 prior to the recruitment of Swi6 [13, 24, 37]; however, it is not known how these establishment factors recruit Clr4 to the heterochromatin regions. There may be a common mechanism of the Clr4 recruitment. Our analyses also demonstrated that the RNAi-RITS pathway is actually involved in the establishment of Swi6 heterochromatin at the cenH-homologous region of the subtelomeres. Although previous work showed that the components of RNAi-RITS are localized at the

Current Biology 1818

telomeres [13, 15, 24, 25], it was unclear whether the RNAi-RITS pathway was directly involved in the formation of Swi6 heterochromatin at telomeres. Indeed, a single deletion of a component of RNAi-RITS does not cause a significant defect in the Swi6 enrichment at the telomeres. In this study, however, we have found that deletion of a component of RNAi-RITS causes severe defects in telomere silencing and Swi6 localization only when it was combined with deletions of taz1+ and the distal region of TAS (Type A chromosome). Furthermore, other lines of our analyses indicated that the RNAi-RITS pathway is directly involved in the establishment of Swi6 heterochromatin at the locus of the telomeric DNA helicase gene. In fact, siRNAs corresponding to the telomeric cenH-like sequences that are associated with RITS have been isolated recently [25]. Thus, the fission-yeast telomere has at least two factors for the establishment of Swi6 heterochromatin, namely, Taz1 and RNAi-RITS. Additionally, unknown factors probably act at the distal region of the TAS to establish Swi6 heterochromatin. The severe defect in the Swi6 localization was observed only when all these factors are deleted (the Type A strain in the absence of Taz1 and RNAi-RITS, or the Type B strain), suggesting that they independently establish Swi6 heterochromatin. This work, along with previous studies [7, 8], suggests that the three constitutive heterochromatin regions, namely, the telomeres, the centromeres, and the silent mat locus, utilize the common RNAi-RITS pathway for establishing heterochromatin in fission yeast. These regions all contain the dh repeat. Importantly, the telomeric DNA helicase ORF contains the partial cenH (dh)-homologous sequences in frame. It is possible that this ORF is the original form of the DNA template for siRNA, and it has been duplicated and translocated to other heterochromatin regions via insertion and mutation of the sequence in the course of evolution. The RNAi machinery is also required for heterochromatin formation in Drosophila and vertebrate cells [38, 39]; however, it remains unknown whether it regulates the formation of telomere heterochromatin in those species. Therefore, the RNAi machinery used for heterochromatin formation may be conserved from yeast to human. Further investigation is required to clarify this matter. At the silent mat locus of fission yeast, Atf1/Pcr1 and RNAi-RITS establish Swi6 heterochromatin in redundant pathways [22]. Telomere has acquired a similar system, namely, the combination of a DNA binding protein (Taz1) and RNAi-RITS. Why is this kind of backup system required? In fission yeast, the telomeric repeats can be easily lost by the mutations in various genes, such as trt1, pot1, or rad3/tel1 [40–43]. Thus, telomeric DNA is relatively unstable compared with other parts of the chromosome. In this regard, the RNAi-RITS pathway may perform the important role of establishing telomere heterochromatin. On the other hand, a chromosome sometimes loses a TAS by accident (e.g., minichromosome), and only the telomeric repeats exist at the chromosome end. In this case, Taz1 is responsible for the establishment of Swi6 heterochromatin at the telomeres. Therefore, those two independent systems are advantageous for the maintenance of stable telo-

mere heterochromatin. Additionally, those two systems can prevent the complete destruction of Swi6 heterochromatin, when a mutation in the genes of the establishment factors is introduced. However, it remains unknown what the centromere-specific factor for the establishment of Swi6 heterochromatin is. Candidate factors are the CENP-B homologs that bind to the dg and dh repeats of the centromeres and are involved in heterochromatin formation, although their localization seems to be not centromere specific [44] (Figure 7B). Supplemental Data Supplemental Data include one supplemental figure and Supplemental Experimental Procedures and are available at http:// www.current-biology.com/cgi/content/full/15/20/1808/DC1/. Acknowledgments We are grateful to R.C. Allshire, S.I.S. Grewal, M. Yanagida, O. Niwa, J. Nakayama, P. Hentges, A. Carr, K. Ishii, K. Ohta, and M. Yamamoto for materials; M. Tamura, T. Naito, A. Matsuura, T. Miyoshi, and T. Mikawa for technical assistance; P. Russell and R. Sugiura for comments on the manuscript; and Y. Chikashige and Y. Murakami for discussion. J.K. was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology. F.I. was supported by a COE Grant, Grants-in-Aid for Cancer Research, and Grants-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology.

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