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. 2008 Apr;28(7):2154-66.
doi: 10.1128/MCB.01637-07. Epub 2008 Jan 22.

Chp1-Tas3 interaction is required to recruit RITS to fission yeast centromeres and for maintenance of centromeric heterochromatin

Jennifer L Debeauchamp  1 , Arian Moses, Victoria J P Noffsinger, Dagny L Ulrich, Godwin Job, Aaron M Kosinski, Janet F Partridge
Affiliations

Chp1-Tas3 interaction is required to recruit RITS to fission yeast centromeres and for maintenance of centromeric heterochromatin

Jennifer L Debeauchamp et al. Mol Cell Biol. 2008 Apr.

Abstract

The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3(Delta)(10-24)) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.

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Figures

FIG. 1.
FIG. 1.
Association between Tas3 and Chp1 is dependent on residues 10 to 24 of Tas3. (A) Summary of two-hybrid interaction data for the binding of truncated Tas3 proteins to full-length Chp1. (B) Two-hybrid analysis of Chp1-GBD or GBD alone (V) binding to Tas3-GAD or GAD alone (V). Growth on synthetic media lacking leucine and tryptophan (SD-L-T) selects for the GAD and GBD vectors, and growth on synthetic media lacking leucine, tryptophan, histidine, and adenine (SD-L-T-H-A) plus X-α-Gal demonstrates the interaction between GAD and GBD fusion proteins. (C) Western blot analysis of the steady-state expression levels of Tas3-GAD and Tas3Δ10-24-GAD fusion proteins in AH109 budding yeast compared to that of Cdc28 as a loading control.
FIG. 2.
FIG. 2.
Tas3Δ10-24-TAP cannot associate with Chp1 but coimmunoprecipitates Flag3-Ago1 from fission yeast whole-cell extracts. (A) Western blot analysis of the tas3-TAP and tas3Δ10-24-TAP alleles in an otherwise wild-type or chp1 null background relative to the expression of the α-tubulin loading control in fission yeast whole-cell extracts. The strains used for this analysis were PY 1064, PY 1210, and PY 1571. (B) Chp1-6xmyc coimmunoprecipitates with Tas3-TAP but not Tas3Δ10-24-TAP from fission yeast. Whole-cell extracts (input) were prepared from fission yeast expressing Chp1-myc6 and either Tas3-TAP or Tas3Δ10-24-TAP. Following IgG Sepharose immunoprecipitation of TAP-tagged proteins, immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide electrophoresis and were subjected to Western blotting with anti-myc antibodies to detect the presence of Chp1-myc6. The strains used for this analysis were PY 1064, PY 1080, and PY 1621. (C) Immunopurification of Tas3Δ10-24-TAP complexes from cells coexpressing chp1-myc6 and Flag3-ago1 alleles reveals the copurification of Ago1 but not Chp1 with Tas3 lacking residues 10 to 24. Immunoprecipitates were subjected to Western blotting with antibodies against the myc, Flag, and TAP (IgG) tags. The strains used were PY 1064, PY 1813, PY 1849, and PY 1908.
FIG. 3.
FIG. 3.
Tas3-Chp1 interaction is required for the maintenance of the silencing of centromeric heterochromatin. (A) Cartoon illustrating the layout of centromere 1 and the position of the cen::ura4+ reporter transgene used in these analyses [at otr1R(dg-glu)SphI::ura4+]. The lower panel is a magnification of the otr1R repeats, highlighting the position of sites A and B in dh and dg, respectively, that are monitored in real-time PCR assays, the probes used for the siRNA analyses from dg and dh (si dg and si dh, respectively), and the position of the nearest gene (rad50+) at the right of centromere 1. Some restriction enzyme sites are marked (N, NcoI; S, SphI; H, HindIII; and X, XhoI). Vertical bars within imr1 (innermost repeat) sequences represent clustered tRNA genes. (B) Comparative growth assay of serially diluted strains bearing the centromeric cen::ura4+ marker within the dg region of centromere 1 or a euchromatic insertion of ura4+ (R.Int::ura4+) and assessed for growth on PMG complete medium, PMG medium lacking uracil (−URA), or PMG complete medium supplemented with 5-FOA (FOA). Cells were wild type (WT), were null for tas3 (tas3Δ), or bore genomic tas3-TAP or tas3Δ10-24-TAP alleles. The strains used were PY 14, PY 30, PY 1402, PY 1640, and PY 1620. (C) Real-time PCR analysis of the relative transcription of centromeric sequences at position A within the dh repeat (see panel A) compared to that of adh1 in random primed cDNA samples generated from the indicated strains. Ratios of centromeric to adh1 transcript levels were normalized to 1 for wild-type strains (WT) and represent mean values for duplicate biological samples, with error bars representing the standard errors of the means. The strains used for this analysis were PY 42, PY 1798, PY 901, PY 938, PY 90, PY 1064, and PY 1571. (D) Analysis of relative transcript levels from centromeric dg sequences at position B within the dg repeat (see panel A) in the RNA samples analyzed in panel C. (E) siRNAs are not produced in Tas3Δ10-24 mutant cells. Small RNAs purified from total cellular RNA were hybridized with dh centromeric probes to reveal siRNAs from the dh region of the centromere, and to probes directed against the small RNA, SnoR69 as the loading control. The strains used were PY 1064, PY 1571, PY 1550, and PY 938. (F) Small RNAs purified from total RNA were analyzed with a dg centromeric probe to reveal siRNAs from the dg region of the centromere, and to SnoR69 as the loading control. The strains used were PY 1064, PY 1571, and PY 938. Excess lanes were removed from the image of the blot, as indicated by the black bar.
FIG. 4.
FIG. 4.
ChIP analysis of the centromeric association of heterochromatin components in mutant cell backgrounds. (A) Real-time PCR analysis of chromatin immunoprecipitated with anti-Ago1 antibody to assess Ago1's association with the outer repeats of the centromere (site A in dh) relative to that of an adh1 euchromatic control. The strains used were PY 1064, PY 1571, and PY 901. (B) ChIP analysis of the association of TAP-tagged proteins with centromeric repeats relative to association at adh1 using IgG Sepharose beads. The strains used were PY 1064, PY 1571, and PY 42. (C) Real-time PCR analysis of ChIP with anti-Chp1 antibodies to assess Chp1's association with dh sequences relative to association with adh1 in mutant backgrounds. Values are shown as the change (n-fold) in enrichment above that of the association of Chp1 in a chp1 null background. The strains analyzed in panels C and D were PY 42, PY 1798, PY 901, PY 938, PY 90, PY 1064, and PY 1571. (D) Real-time PCR analysis of ChIP with anti-H3K9Me2 antibody to assess H3K9Me2 enrichment at centromeric sequences within dh (site A) relative to that of an adh1 euchromatic control. Immunoprecipitation was performed with anti-H3K9Me2 antibody on a clr4 null strain (second lane), and this served as a normalization control for the other samples. (E) Real-time PCR analyses of ChIPs with anti-H3K9Me2 antibody to assess the enrichment of centromeric dg sequences (site B) in the samples analyzed in panel D. (F) ChIP analysis of the association of Rdp1-Flag3 with centromeric repeats relative to association with adh1. Strains were tested for the association of Rdp1 with centromeric dh sequences relative to that at adh1 by ChIP with anti-Flag antibody. The strains used in the analysis were PY 2028, PY 3383, and PY 3249.
FIG. 5.
FIG. 5.
Tas3Δ10-24-TAP impacts the establishment, but not maintenance, of heterochromatin at the mating-type locus. (A) Cartoon of the mat2/mat3 locus showing the localization of the ade6+ reporter gene inserted at mat3 (EcoRV). Shown are serial-dilution growth assays of cells that were wild type for ade6+ or that carried a wild-type ade6 allele at mat3 (mat3M::ade6+) in different genetic backgrounds and spotted onto PMG complete medium supplemented with adenine (Full Ade) or without adenine (No Ade). The strains analyzed were PY 12, PY 261, PY 1157, PY 260, PY 1127, PY 2270, PY 2271, and PY 2272. (B) Serial dilution assay of cells bearing the mat3M::ade6+ reporter spotted onto PMG complete medium, PMG medium supplemented with a low level of adenine (12% Ade), or medium containing no adenine (No Ade). The strains analyzed were PY 261, PY 2271, PY 2272, PY 1157, PY 2829, PY 2830, PY 2831, and PY 2832, with the last four being clr4 null strains into which clr4+ was reintegrated to allow the monitoring of the establishment of heterochromatin.
FIG. 6.
FIG. 6.
Chp1 and Tas3, but not the RITS, are required for the maintenance of the silencing of telomeric transcripts. (A) Cartoon showing the location of subtelomeric tlh+ genes (black) and the position of the PCR amplicon A within the helicase domain (gray), which is separate from the centromere homology region (striped). Also shown is the real-time PCR analysis of the accumulation of subtelomeric transcripts (measured at position A in tlh+ genes) relative to that of adh1+ control euchromatic transcripts in cDNA prepared from strains of different genetic backgrounds. The strains analyzed were PY 42, PY 1798, PY 901, PY 938, PY 90, PY 1064, and PY 1571. (B) Real-time PCR analysis of tlh transcript accumulation in double-null mutants of chp1 and tas3, performed as described for panel A, using strains PY 42, PY 1798, PY 938, PY 90, PY 2610, and PY 2613. (C) ChIP analysis of Chp1 association with telomeric sites relative to that of adh1, performed with anti-Chp1 antibodies and measured by real-time PCR. The same strains that were used for panel A were used for this analysis. (D) ChIP analysis of H3K9Me2 levels at subtelomeric sites (designated A) relative to levels at adh1 in PY 1064, PY 1571, and PY 1798. (E) ChIP analysis of Tas3-TAP and Tas3Δ10-24-TAP recruitment to tlh sequences relative to levels at adh1, performed with anti-TAP antibodies and measured by real-time PCR. Experiments were performed on strains lacking a TAP tag on Tas3 (tas3+), tas3-TAP, and tas3Δ10-24-TAP, each in both chp1+ and chp1Δ backgrounds (PY 42, PY 1064, PY 1571, PY 2208, PY 2174, and PY 90). (F) The Chp1-Tas3 complex is required for the establishment of telomeric heterochromatin. Shown is a real-time PCR analysis of tlh transcript accumulation compared to that of the euchromatic control adh1 transcripts in cDNA generated from PY 42, PY 1798, PY 3435, PY 3436, PY 3438, PY 3439, PY 3441, PY 3442, PY 3444, and PY 3445. (G) Real-time PCR analysis of subtelomeric transcripts in strains expressing untagged Tas3Δ10-24 demonstrates tlh transcript accumulation. tlh transcripts were compared to euchromatic adh1 control transcripts in cDNA generated from strains PY 42, PY 1798, PY 938, PY 90, and PY 3506. (H) Real-time PCR analysis of transcripts from the dh region of the centromeric repeats relative to those of adh1 control transcripts in the samples analyzed in panel G. wt, wild type.
FIG. 7.
FIG. 7.
Model for the role of Chp1-Tas3 interaction in the maintenance of centromeric heterochromatin. We propose the following steps. (1) Low levels of Clr4 are recruited to centromeres via an RNAi-independent mechanism and provide H3K9Me2. (2) The H3K9Me2 is bound by Chp1 in the context of RITS (green). (3) The centromeric association of RITS recruits Rdp1 and RDRC (brown). (4) Rdp1 uses the single-stranded pre-siRNA polymerase II transcript as the template for the synthesis of dsRNA. (5) Dcr1 cleaves the dsRNA to generate siRNAs. (6) The siRNAs eventually are loaded onto Ago1 in RITS and allow the Ago1-mediated cleavage of nascent centromeric transcripts, which contributes to centromeric silencing (not shown). (7) The centromeric association of RITS and RDRC facilitates the recruitment of further Clr4, which promotes the accumulation of high levels of H3K9Me2 at centromeres, enhancing the recruitment of RITS and RDRC in an RNAi-dependent positive feedback loop to allow the robust assembly of centromeric heterochromatin. In strains bearing the Tas3Δ10-24 mutant (lower panel), this RNAi-dependent positive feedback loop is disengaged, since the loss of the Chp1-Tas3 association disrupts the recruitment of RITS and RDRC to centromeres, causing a failure in the production of siRNAs and the recruitment of only low levels of H3K9Me2 to centromeres, levels that are insufficient to support the maintenance of centromeric heterochromatin.
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