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. 2018 Aug 23;7:e37911. doi: 10.7554/eLife.37911

Figure 1. Two copies of Cse4 within a nucleosome are required for yeast viability.

(A) Design of an asymmetric Cse4 interface. Secondary structure and sequence comparison of the Cse4 (white) and H3 (blue) histone-fold domains are shown at the top. Non-identical residues are shaded, and red residues indicate the asymmetric alterations (Ichikawa et al., 2017). The Chimera construct includes H3 residues within the context of Cse4 (G196S, L198F, H200D, L204A, L206I, I224L), and this was used to create the asymmetric Cse4X (G196S, L198F, H200D, L204A, L206I, L220A, I224V) and Cse4Y (G196S, L198F, H200D, L203I, L204W, L206I) proteins. The asymmetric H3X (L126A, L130V) and H3Y (L109I, A110W, L130I) proteins are illustrated for comparison. (B) Genetic analysis of heterodimeric Cse4X/Cse4Y pairs. Neither Cse4X alone nor Cse4Y alone support growth. Images show growth of yeast cells upon 5-FOA selection against a URA3-containing plasmid carrying cse4-107 (Chen et al., 2000), comparing strains expressing wild-type Cse4, Chimera, Cse4X alone, Cse4Y alone, both Cse4X and Cse4Y, or no Cse4 (empty vector). Colonies were picked from selective media and patched on SC-TrpLeu and FOA plates simultaneously (left panels). Three independent transformants for each strain were grown for 3 days on SC-TrpLeu plates or 7 days on FOA plates. Right panels show replica plating controls to ensure adequate numbers of cells were analyzed. The primary SC-TrpLeu plate (upper left) was replica plated onto SC-TrpLeu as a positive control for cell transfer and onto FOA to test for Cse4 function; both replica plates were incubated for three days. Note that no growth on FOA was observed for any of the Cse4X alone and Cse4Y alone isolates. (C) Growth assay for the indicated strains under stress conditions. (Top row) Serial dilutions of the indicated strains were plated on YPD plates and were incubated at 30°C, 34°C or 37°C for 2 days. (Bottom row) Cells were plated on YPD, YPD +0.2% DMSO or YPD +10 μg/ml benomyl with 0.2% DMSO and were incubated at 30°C for 2 days. Yeast carrying Chimera and Cse4 X + Y nucleosomes grow slower than wild-type (Cse4 WT) at 37°C, and both strains are slightly sensitive to benomyl treatment relative to wild-type. Strains analyzed were: Cse4 WT (PKY5230), Chimera (PKY5232), Cse4 X + Y (PKY5234).

Figure 1.

Figure 1—figure supplement 1. Simple insertion of asymmetric residues into Cse4 results in non-functional proteins.

Figure 1—figure supplement 1.

A) Design of Cse4X’ and Cse4Y’. As in Figure 1, secondary structure and sequence comparison of the Cse4 (white) and H3 (blue) histone-fold domains are shown. Asymmetric mutation sites (red, Ichikawa et al., 2017]) of Cse4X’ (L204A, L220A, I224V) and Cse4Y’ (L203I, L204W) are mapped on the secondary structure. (B) Genetic analysis of Cse4X’+Cse4Y’ pairs. Cse4X’ and Cse4Y’ cannot support growth in the absence of Cse4-107. Images show growth of three independent transformants for each strain under FOA selection as in Figure 1B.
Figure 1—figure supplement 2. Analysis of paired electrostatic residues within the Cse4 dimerization region.

Figure 1—figure supplement 2.

(A) Design of Cse4D/Cse4H and Cse4D’/Cse4H’. As in Figure 1, secondary structure and sequence comparison of the Cse4 (white) and H3 (blue) histone-fold domains are shown. Mutation sites of Chimera (G196S, L198F, H200D, L204A, L206I, I224L), Cse4D (G196S, L198F, H200D, L204D, L206I), Cse4H (G196S, L198F, H200D, L204A, L206I, I224H), Cse4D’ (L204D), Cse4H’ (I224H), H3D (A110D) and H3H (L130H) are mapped on the secondary structure. Red residues indicate the electrostatic mutations (Zhou et al., 2017).(B) Genetic analysis of Cse4D/Cse4H and Cse4D’/Cse4H’ pairs. Yeast cells expressing Cse4H alone or Cse4H’ alone support growth in the absence of Cse4-107. As in Figure 1B, images show growth of three independent transformants for each strain during FOA selection against a URA3-marked plasmid carrying cse4-107.
Figure 1—figure supplement 3. Genetic analysis of H3D/H3H and H3X/H3Y pairs.

Figure 1—figure supplement 3.

Yeast cells expressing H3H alone support growth in the absence of wild-type H3. Images show growth of yeast carrying wild-type H3 on a URA3-marked plasmid as well as either H3D alone, H3H alone, both H3D and H3H, H3X alone, H3Y alone, or both H3X and H3Y. All test plasmids were introduced into strain LHT001 (the histone shuffle strain used in (Zhou et al., 2017), and three independent transformants for each test strain were grown on 5-FOA to select against the URA3 shuffle plasmid. Plate layout is shown in the upper left. Auxotrophic makers of each plasmid (LEU2 or TRP1) are shown in parenthesis.
Figure 1—figure supplement 4. Temperature-sensitive growth assays.

Figure 1—figure supplement 4.

Yeast cells expressing H3D/H3H or H3X/H3Y were streaked on YPD plates and incubated at 30°C for 2 days, or 37°C for 3 days. Both asymmetric H3 pairs caused severe growth defects at 37°C. Plate layout is shown in the upper left. PKY4704, which is expressing H3X/H3Y (in strain PKY4701 (Ichikawa et al., 2017]) was used as a positive control for temperature sensitivity at 37°C. All other test strains expressing H3D/H3H or H3X/H3Y were derived from LHT001 as described in Figure 1—figure supplement 3.
Figure 1—figure supplement 5. Biochemical analysis of asymmetric Cse4 nucleosome formation in vivo.

Figure 1—figure supplement 5.

(A) Schematic for in vivo biochemical analysis of Cse4X-Y dimerization. Yeast strains expressed 3xFLAG-tagged Cse4X or 3xFLAG-tagged Cse4Y, along with 3xV5-tagged Cse4X and 3x HA-tagged Cse4Y, as indicated. Cse4 nucleosomes were solubilized by MNase digestion, immunoprecipitated with anti-FLAG agarose beads, and analyzed by immunoblotting. (B) Immunoblot analysis. For each strain, the left lane shows total soluble Cse4 molecules (Input), and the right lane shows co-precipitated Cse4 species (Bound). The same blot was probed sequentially with indicated antibodies (V5, HA or FLAG).