Volume 26, Issue 4, Pages 593-602 (May 2007) Functional Separation of the Requirements for Establishment and Maintenance of Centromeric Heterochromatin  Janet F. Partridge, Jennifer L. DeBeauchamp, Aaron M. Kosinski, Dagny L. Ulrich, Michael J. Hadler, Victoria J.P. Noffsinger  Molecular Cell  Volume 26, Issue 4, Pages 593-602 (May 2007) DOI: 10.1016/j.molcel.2007.05.004 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Ago1 Associates with Tas3 through a Conserved GW-Rich Motif (A) Tas3-TAP coimmunoprecipitates Ago1 independent of its association with Chp1, but Chp1-TAP cannot immunoprecipitate Ago1 from tas3-null extracts. Cell extracts were prepared from cells of the indicated genotypes, and IgG Sepharose was used to purify Tas3-TAP (upper panel) or Chp1-TAP complexes (lower panel). Immunoblotting with anti-Flag antibodies revealed that Tas3 binds Ago1 independently of Chp1, whereas association of Chp1-TAP with Ago1 is dependent on Tas3. Strains used were PY42, 1064, 1732, 1784, and 1832 (upper panel) and PY42, 736, 1732, 1834, and 2104 (lower panel). (B) Chp1-TAP coimmunoprecipitates Ago1 from cells expressing Tas3 1–380, but not Tas3 1–282. Cell lysates were prepared from tas3-null strains transformed with plasmids expressing full-length Tas3 (1–549), truncations of Tas3, or empty vector (−) and expressing Chp1-TAP and 3×Flag-Ago1. IgG Sepharose purification of Chp1-TAP complexes revealed that residues 1–380 of Tas3 are sufficient for the interaction between Tas3 and Ago1. Plasmids were transformed into PY2104. (C) Tas3 encompasses a conserved GW-rich Argonaute interaction motif. Pile-up analysis of Tas3 with TNRC6B proteins from different species. Sequence identity is highlighted by asterisks, and mutations generated in Tas3 are marked beneath the text. Black, no impact on centromeric function; red, residues that when mutated impacted Tas3 function. Residues shown are 201–311 of Tas3, 755–873 of Chimp TNRC6B (XM_515145), 897–1016 dog TNRC6B (XM_538361), 932–1051 mouse TNRC6B (NM_144812), and 898–1017 human TNRC6B (XM_039385). (D) The mutant Tas3WG-TAP protein does not coimmunoprecipitate Ago1 from cell lysates. Whole-cell extracts were prepared from cells expressing genomic 3×Flag-Ago1, nontagged Tas3, Tas3-TAP, or Tas3WG-TAP proteins, and IgG Sepharose immunoprecipitates were probed with anti-Flag and anti-IgG antibodies after electrophoresis and blotting. Strains used were PY1813, 1849, and 2323. Molecular Cell 2007 26, 593-602DOI: (10.1016/j.molcel.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Tas3WG Cells Show No Defect in Maintenance of Centromeric Heterochromatin, but Maintenance of Centromeric Heterochromatin in These Cells Relies on Ago1-siRNA Interaction (A) Comparative growth assay of serially diluted cells bearing the cen::ura4+ transgene and plated on PMG complete medium, PMG medium lacking uracil, or PMG complete medium supplemented with 5-FOA. Strains are wild-type tas3+, tas3 null (tas3Δ), or expressed wild-type or mutant Tas3-TAP fusion proteins from the endogenous locus (PY30, 1640, 1402, 2310, and 2311). (B) Real-time PCR analysis of centromeric sequence representation (site A in dh region of the centromere) compared with adh1 in cDNA generated by random priming from RNA derived from Tas3-TAP, Tas3WG-TAP, dcr1-, tas3-, or ago1-null cells (PY901, 1550, 938, 1064, and 2267). Data are represented as mean ± SEM. (C) ChIP analysis of (upper panel) Ago1 association with centromeric repeat sequences (site A in dh) in Tas3-TAP and Tas3WG-TAP strains, normalized to the euchromatic adh1 control sequence, (middle panel) Chp1 association, and (lower panel) Tas3 association. Strains used were PY1064, 2267, 901, 90, and 42. Data are represented as mean ± SEM. (D) Small RNAs purified from Tas3WG cells were hybridized with centromeric probes to reveal centromeric siRNAs and to the small RNA snoR69 as a loading control. Strains used were PY1064, 2267, and 938. (E) Real-time PCR analysis of centromeric transcript levels (site A in dh of the outer repeat) compared with adh1 transcripts in cDNA generated by random priming from RNA derived from cells bearing combinations of mutant ago1 alleles (F276A-ago1) and tas3-alleles (tas3WG-TAP). Strains used for this analysis were PY2781, 2780, 2776, 2778, and 1402. Data are represented as mean ± SEM. (F) Small RNAs purified from tas3WG and F276A-ago1 mutant cells were hybridized with centromeric probes to reveal centromeric siRNAs and to the small RNA snoR69 as a loading control. The same strains were analyzed as in Figure 2E. Molecular Cell 2007 26, 593-602DOI: (10.1016/j.molcel.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Tas3WG Cells Are Deficient in the Establishment of Centromeric Heterochromatin (A) Comparative growth assay of serially diluted strains bearing the dg cen::ura4 transgene and assessed for growth on PMG (complete), PMG medium lacking uracil (−Ura), or PMG medium supplemented with 5-FOA (+ FOA). Cells were wild-type for Clr4 (WT) or were null for clr4 (clr4Δ). clr4+ was reintroduced by integration into the clr4 locus (clr4Δ to clr4+). Strains used in this analysis were PY2756, 2646, 2678, 2679, 2648, 2680, 2681, and 2682. (B) Real-time PCR analysis of centromeric transcripts arising from the dh region (A), relative to adh1 transcripts in cDNA generated by random priming of total RNA. Strains used were PY2755, 2759, 2678, 2680, and 2756, and these were used also in (C) and (D). Data are represented as mean ± SEM. (C) ChIP analyses of (upper panel) Ago1 association, (middle panel) H3K9Me2, and (lower panel) Chp1 association with the dh region of the centromere relative to adh1, measured by real-time PCR. Data are represented as mean ± SEM. (D) Small RNAs purified from total cellular RNA were hybridized with centromeric probes to reveal centromeric siRNAs and to the small RNA snoR69 as the loading control. Molecular Cell 2007 26, 593-602DOI: (10.1016/j.molcel.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Tas3WG Cells Can Assemble Centromeric Heterochromatin after dcr1+ Reconstitution (A) ChIP analysis of H3K9Me2 at the dh region of the centromere relative to adh1, measured by real-time PCR. Strains used were PY42, 1150, and 1798. Data represent the mean of three experiments ± SEM. (B) Analysis of small RNAs purified from total RNA and blotted for centromeric siRNAs or the loading control snoR69. Strains were transformed with ura4+ marked plasmids and were grown in PMG-uracil media. Strains used were PY1064 (transformed with empty vector), 3019 (transformed with empty vector), 3017 (transformed with pREP2-dcr1+), and 3019 (transformed with pREP2-dcr1+). Similar data were obtained for four independent pREP2-dcr1+ transformants for 3017 and 3019. The same strains and growth regimen were used for transcript analyses in (C) and (D). Excess lanes were removed from the image of the blot, as indicated by the black bar. (C) Real-time transcript analysis of centromeric dh transcripts relative to adh1 transcripts. Data represent mean data obtained for four or five transformants for reintroduction of dcr1+, and two isolates for controls, and are represented as ± SEM. (D) Real-time PCR analysis of centromeric dg transcripts relative to adh1 transcripts for the same RNA samples as used in (C). (E) Model for the role of RITS in the maintenance and establishment of centromeric heterochromatin. Black wavy line represents nascent centromeric pre-siRNA transcripts, and gray triangles represent siRNA. Molecular Cell 2007 26, 593-602DOI: (10.1016/j.molcel.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions