1. Telomere Binding Protein Taz1 Establishes Swi6 Heterochromatin Independently of RNAi at Telomeres 
Junko Kanoh, Mahito Sadaie, Takeshi Urano, Fuyuki Ishikawa 
Current Biology 
Volume 15, Issue 20, Pages (October 2005)
DOI: /j.cub
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2. 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 swi6Δ. Error bars indicate the standard error from multiple independent experiments.
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3. 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 ∼300 bp long, the TAS is ∼10 kb long, and the lambda DNA is ∼11 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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4. 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 taz1Δ. 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.
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5. 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 K9-methylated 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 ∼14 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.
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6. 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 ∼11 and ∼33 kb from the chromosome end was examined. Serial cell dilutions of the wild-type, swi6Δ, dcr1Δ, and chp1Δ 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. ChIP analyses of Swi6 localization at the TAS (∼11kb 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 (∼14 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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7. 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 ∼9 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 ∼11 kb from the original chromosome end. Error bars indicate the standard error from multiple independent experiments.
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8. Figure 7
Model of the Establishment of Heterochromatin in Fission Yeast
(A) Establishment and spreading of Swi6 heterochromatin at telomeres. Taz1 and RNAi-RITS 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.
Current Biology  , DOI: ( /j.cub )
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