The NASP-Related Protein Sim3 Is Required for Central Core Silencing at Centromeres and Normal Chromosome Segregation
(A) S. pombe centromeres consist of a central core domain surrounded by outer repeat regions. The arg3⁺ insertion at cnt1 allows monitoring of central core silencing (Pidoux et al., 2003).
(B) Left, serial dilution to monitor silencing at central core (cnt1:arg3⁺), outer repeat heterochromatin (otr2:ura4⁺), and telomeres (tel1L:his3⁺), assayed by growth on indicated media. sim3-143, sim3-205, cnp1-76, and mis6-302 mutants specifically alleviate central core silencing. Outer repeat and telomeric silencing is alleviated in the heterochromatin mutant rik1Δ. Right, serial dilution assay to assess growth and viability of sim3 mutants compared to wild-type at 25°C and 36°C on YES + phloxine B; darker pink colonies contain more dead cells. Strains are FY3027, 6154, 5496, 4462, 5691, and 3606.
(C) Chromosome segregation defects in sim3-143. Wild-type and sim3-143 cells were shifted to 36°C for 6 hr before fixation and immunolocalization with antibodies to α-tubulin (microtubules; green) and DAPI staining (DNA; red). Scale bar, 5 μm.
(D) Quantification of chromosome segregation patterns. Numbers are percentages of each pattern in early (short spindles) and late mitosis (elongated spindles) in cultures grown at 36°C (6 hr).

CENP-A^Cnp1 Levels at Centromeres Are Reduced in sim3 Mutants
(A) CENP-A^Cnp1 localization in wild-type and sim3 strains at 25°C and 36°C. Strains were grown at 25°C or shifted to 36°C for 6 hr before fixation and processing for immunolocalization with anti-CENP-A^Cnp1 (green), anti-Sad1 (red), and DAPI (blue). Scale bar, 10 μm.
(B) Top, ChIP of CENP-A^Cnp1 in wild-type and sim3 mutants at 25°C and 36°C (6 hr). Immunoprecipitated DNA was analyzed by multiplex PCR. cnt1 or imr1 enrichment is measured relative to the fbp1 euchromatic control and normalized to the input (T, Total) PCR. Bottom, ChIP of histone H3 in wild-type and sim3 mutants at 25°C and 36°C. The kinetics of fixation differ at 25°C and 36°C, and so ChIPs at the two temperatures are not directly comparable.
(C) ChIP of CENP-A^Cnp1 in wild-type and sim3 mutants at 25°C and 36°C. In a separate experiment from (B), the level of cnt1 and act1 DNA in the input and anti- CENP-A^Cnp1-immunoprecipitated chromatin was determined by quantitative real-time PCR.

Association of CENP-A^Cnp1 and H3 with Sim3
(A) Sim3-GFP and myc-CENP-A^Cnp1 coimmunoprecipitate. Extracts were prepared from cells in which Cnp1 and Sim3 were untagged (FY1645) or which expressed Sim3-GFP (FY6322), or myc-CENP-A^Cnp1 (FY5927), or both Sim3-GFP and myc-CENP-A^Cnp1 (FY6374). Immunoprecipitations were performed with sheep anti-GFP or mouse anti-myc (9E10). Immunoprecipitates (IP) and whole-cell extracts (WCE) were analyzed by western blot with rabbit anti-GFP or rabbit anti-myc antibodies as indicated. Positions of Sim3-GFP and Myc-CENP-A^Cnp1 proteins and molecular weight standards are indicated. Long exposures of two panels are shown on the right for comparison.
(B) GFP-CENP-A^Cnp1 and histone H3-GFP coimmunoprecipitate with Sim3. Extracts were prepared from strains expressing no tagged proteins (FY1645), GFP-CENP-A^Cnp1 (FY5205), histone H3-GFP (FY6443, a gift from Mohan Balasubramanian), or Sim3-GFP (FY6322). Immunoprecipitations were performed with sheep anti-GFP or rabbit anti-Sim3. IPs and WCEs were analyzed by western blot with rabbit anti-Sim3 or rabbit anti-GFP antibodies as indicated. Positions of Sim3, Sim3-GFP, H3-GFP, and GFP-CENP-A^Cnp1 proteins and molecular weight standards are indicated. Different length exposures are shown for different panels to allow relevant bands to be seen clearly.
(C) Reciprocal coimmunoprecipitations from extracts of cells expressing Sim3-GFP and myc-CENP-A^Cnp1 (FY6374) or Sim3-143-GFP and myc-CENP-A^Cnp1 (FY6368/9), grown at 25°C or 36°C (6 hr). Antibodies as in (A).
(D) Sim3 and CENP-A^Cnp1 interact in vitro. Left, ³⁵S-labeled Sim3 produced by in vitro transcription and translation was incubated with GST fusion proteins, and pull downs were analyzed by SDS-PAGE and fluorography. ³⁵S-labeled Sim3 (1/5th) was run in the left lane. Right, binding of mutant ³⁵S-labeled Sim3-143 and Sim3-205 to GST-CENP-A^Cnp1 was compared to wild-type in the in vitro binding assay.

Sim3 Aids the Deposition of GFP-CENP-A^Cnp1 during S and G2
(A) Wild-type and cdc10-129 mutant (G1 arrest) cells were incubated at 36°C for 3 hr followed by induction of inv1-GFP-CENP-A^Cnp1 for 60 min in sucrose medium (4 hr total at 36°C). Cells were fixed and analyzed for presence of GFP-CENP-A^Cnp1 spot by fluorescence microscopy.
(B) Wild-type, sim3-143, sim3-205, and mis6-302 cells were arrested in S phase by the addition of 25 mM hydroxyurea (HU) for 4 hr at 25°C, followed by induction of inv1-GFP-CENP-A^Cnp1 in sucrose media containing HU for a further 1 hr (5 hr total). Cells were analyzed as in (A).
(C) Incorporation of newly synthesized GFP-CENP-A^Cnp1 in cdc25-22 (FY8518), sim3-143 cdc25-22 (FY8717), and sim3-205 cdc25-22 (FY8718) strains at 36°C. Cultures grown at 25°C were shifted to 36°C for 3 hr in repressed conditions, then inv1-GFP-CENP-A^Cnp1 was induced for a further 1 hr at 36°C. Cells were fixed, DAPI stained, and analyzed by fluorescence microscopy for the presence of GFP-CENP-A^Cnp1 signal (n = 200 for each strain). Scale bar, 10 μm.
(D) Model: Sim3 (C shape) acts a classic chaperone, directly binding CENP-A^Cnp1 (white triangle), escorting it to the centromere, and handing it over to centromere-associated chromatin assembly factors (white crescent, X) that incorporate CENP-A^Cnp1 in place of histone H3. CENP-A^Cnp1 and H3 nucleosomes are shown as white cubes and gray cylinders, respectively. Evicted H3 may also be received by Sim3.

Sim3 Has Interrupted TPR Motifs
(A) Motif arrangement in the SHNi-TPR family: four TPR-related motifs (M1–M4) are predicted (see [B]), and a carboxy-terminal motif (CM) also shares similarity.
(B) Global alignment of amphipathic helices in motifs –M4 against TPR motif. S. pombe Sim3 (S.p.) was aligned with related proteins from other species (see Supplemental Data). The secondary structure from TPR1 of PP5 (PDB code 1A17) is shown above the alignment. Motifs M1, M3, and M4 are predicted to be TPR-like motifs in Human NASP. Global alignment shows that M1, M2, M3, and M4 can be structurally aligned with a TPR motif if insertions are allowed between the amphipathic helices. These insertions are sparse in hydrophobic residues and are likely to be unstructured. The hydrophobic residues that define the TPRs are shown as white-on-black boxes. The positions of mutations in Sim3 are highlighted in blue. Residue 2 of all four SHNi-TPR motifs and residue 9 of M3 are highlighted in red.
(C) A model of a four-TPR structure extrapolated from the structure of the three TPRs of PDB code 1fch. Insertions could be accommodated in the links between helices and the links between TPR modules. The Ca and Cb carbons of Residue 2 (M1–M4) and residue 9 of M3 are shown as red spheres. The protein is shown in blue to red graded by sequence number from N to C terminus.

Sim3 Is Distributed throughout the Nucleus
(A) Localization of Sim3 to the nucleus throughout the cell cycle. Immunofluorescence of wild-type cells using affinity-purified anti-Sim3 antibody (red), anti-tubulin (microtubules; green), and DAPI (DNA; blue). Merged images are shown on the left. Scale bar, 10 μm.
(B) Fluorescence images of live interphase cells expressing Sim3-GFP (FY6326) and Sim3-143-GFP (FY6308).
(C) Western analysis using anti-Sim3 antibody to detect levels of Sim3 in wild-type and sim3 mutants at 25°C and 36°C (6 hr) or anti-tubulin as a loading control.

Sim3 Mediates the Incorporation of Newly Synthesized GFP-CENP-A^Cnp1 at Centromeres
(A) Schematic of strain with inducible GFP-CENP-A^Cnp1 expressed from inv1 promoter.
(B) Northern and western time-course analysis showing induction of GFP-CENP-A^Cnp1 transcript and protein from the invertase promoter (inv1) in wild-type (FY8481), sim3-143 (FY8482), and mis6-302 (FY8519) at 25°C. adh1 was used as a loading control for northern analysis. Levels of induced GFP-CENP-A^Cnp1 protein were determined by anti-GFP western analysis, and Bip1 was used as a loading control. R, repressed conditions; times of induction are given in minutes.
(C) Cells grown in repressed conditions (10% glucose) at 25°C were fixed and analyzed by fluorescence microscopy; very few cells showed a GFP-CENP-A^Cnp1 signal (<5%).
(D) Incorporation of newly synthesized GFP-CENP-A^Cnp1 in wild-type, sim3-143, sim3-205, and mis6-302 at 25°C and 36°C. Cultures were grown at 25°C or shifted to 36°C for 5 hr under repressed conditions, then inv1-GFP-CENP-A^Cnp1 was induced by switching to sucrose media for a further hour at either 25°C or 36°C (for details see text). Cells were fixed and analyzed by fluorescence microscopy, and the presence of the characteristic CENP-A^Cnp1 signal was scored for each strain and condition (percentage indicated; n = 200).
(E) Anti-GFP ChIP on wild-type and sim3-143 strains containing inv1-GFP-CENP-A^Cnp1 at 25°C, grown under repressed or induced (1 hr) conditions. ChIPs were analyzed by PCR: cnt1 enrichment is measured relative to the fbp1 euchromatic control and normalized to the input.