1. Chromosomes and Gene Expression
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An asymmetric centromeric nucleosome

  1. Yuichi Ichikawa
  2. Noriko Saitoh
  3. Paul D Kaufman  Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. The Cancer Institute of JFCR, Japan
Research Advance
Cite this article as: eLife 2018;7:e37911 doi: 10.7554/eLife.37911
4 figures and 2 additional files

Figures

Figure 1 with 5 supplements
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).

https://doi.org/10.7554/eLife.37911.002
Figure 1—figure supplement 1
Simple insertion of asymmetric residues into Cse4 results in non-functional proteins.

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.

https://doi.org/10.7554/eLife.37911.003
Figure 1—figure supplement 2
Analysis of paired electrostatic residues within the Cse4 dimerization region.

(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.

https://doi.org/10.7554/eLife.37911.004
Figure 1—figure supplement 3
Genetic analysis of H3D/H3H and H3X/H3Y pairs.

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.

https://doi.org/10.7554/eLife.37911.005
Figure 1—figure supplement 4
Temperature-sensitive growth assays.

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.

https://doi.org/10.7554/eLife.37911.006
Figure 1—figure supplement 5
Biochemical analysis of asymmetric Cse4 nucleosome formation in vivo.

(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).

https://doi.org/10.7554/eLife.37911.007
A single N-terminal domain within each Cse4 nucleosome is sufficient for yeast viability.

(A) The location of the Essential N-terminal Domain (END) is diagrammed relative to the alpha helices within the Cse4 histone-fold domain. The N-terminal 70 amino acids containing END were deleted from each construct, as indicated (Δ70 strains). (B) Viability test of the Δ70 strains. Deletion of both END regions within a nucleosome was lethal, but a single END per Cse4 nucleosome supports growth. As in Figure 1B, images show growth of yeast during selection against a URA3-marked plasmid carrying cse4-107. Three independent transformants expressing the indicated proteins (Cse4 Wild-type, Cse4 Δ70, Chimera, Chimera Δ70, Cse4 X + Cse4 Y, Δ70X + Y, X + Δ70Y or Δ70X + Δ70Y) were grown on 5-FOA plates for 7 days. (C) Growth assay for the indicated strains under stress conditions. Serial dilutions of the indicated strains were plated on YPD or YPD +10 μg/ml benomyl with 0.2% DMSO. YPD plates were incubated at 30°C or 34°C for 3 days. The benomyl plate was incubated at 30°C for 3 days. Strains analyzed were: Cse4 WT (PKY5470), X + Y (PKY5476), Δ70X + Y (PKY5485) and X + Δ70Y (PKY5488).

https://doi.org/10.7554/eLife.37911.008
Effects of asymmetric Cse4 mutations on plasmid loss rates.

Mitotic plasmid stability assay. Test strains bearing plasmid pRS413 (Sikorski and Hieter, 1989), which contains a centromere (CEN6), an autonomously replicating sequence (ARS), and the selectable marker HIS3, were grown in unselective media (YPD) for approximately 10 generations at 30°C. The fraction of plasmid-containing cells was determined by plating identical aliquots on selective and nonselective media. At least four biological replicates were analyzed for each mutant strain. Individual data points, along with average and standard deviation of the plasmid loss rates are graphed on the y-axis. Constructs that resulted in loss rates significantly different than the pseudo wild-type strain are shown in red (p<0.05, Welch’s t test).

https://doi.org/10.7554/eLife.37911.009
Figure 4 with 3 supplements
A single CENP-C interaction region per Cse4 nucleosome supports viability.

(A) As in Figure 1, secondary structure and sequence comparison of the Cse4 (white) and H3 (blue) histone-fold domains are shown. ERS mutations (C-terminus QFI/ERS swap) on each construct are indicated. The altered Cse4 or H3 amino acids are shown above the secondary structure, and the substituted amino acids are shown below. Red residues indicate the asymmetric mutations (Ichikawa et al., 2017). (B) Growth assay for the indicated strains under stress conditions. (Upper panel) For Cse4 nucleosomes built with the Chimera protein, ERS mutations on both Cse4 molecules resulted in severe growth defects. (Lower panel) In the asymmetric Cse4 nucleosome, a single ERS mutation did not cause significant growth defects, even under stress conditions. Serial dilutions of the indicated strains were plated on YPD or YPD +10 μg/ml benomyl with 0.2% DMSO. YPD plates were incubated at 30°C, 34°C or 37°C for 3 days. The benomyl plate was incubated at 30°C for 3 days. Strains analyzed were: Chimera (PKY5473), Chimera-ERS (PKY5494), X + Y (PKY5476), X-ERS +Y ERS (PKY5503), X-ERS +Y (PKY5497) and X + Y ERS (PKY5500).

https://doi.org/10.7554/eLife.37911.010
Figure 4—figure supplement 1
Analysis of additional mutations affecting the CENP-C interaction region.

(A) Cse4 regions important for CENP-C interaction (from the Loop1 to the C-terminus (Xiao et al., 2017) were partially substituted with the analogous residues from H3. The ‘ERS*’ alterations consist of Loop1 alterations (deletion of KDQ residues in Cse4 Loop1, plus a W178F swap mutation) plus the ERS alterations (swap of the C-terminal QFI/ERS residues), as shown. (B) Viability test of the ERS* strains. The ERS* alterations on the homodimeric Cse4/H3 chimera background is lethal, but ERS* on a single half of the asymmetric Cse4 nucleosome supports viability. 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. Note that no growth on FOA was observed for the Chimera-ERS* or X-ERS*+Y-ERS* strains. (C) Growth assay for the indicated strains under stress conditions, as in Figure 4B. Strains analyzed were: X + Y (PKY5476), X-ERS +Y (PKY5497), X + Y ERS (PKY5500), X-ERS +Y ERS (PKY5503), X-ERS*+Y (PKY5509) and X + Y-ERS* (PKY5512).

https://doi.org/10.7554/eLife.37911.011
Figure 4—figure supplement 2
Combination of N- and C-terminal Cse4 alterations.

Combining Δ70 and ERS* mutations, either in cis (mutated on the same Cse4 molecule within a nucleosome) or trans (mutated on different Cse4 molecules within a nucleosome) yielded cells that were initially viable. However, these cis and trans mutant strains displayed severe growth defects under normal growth conditions (YPD, 30°C). Strains with Δ70 and ERS* mutations on both Cse4 molecules within a nucleosome were completely inviable. (A) Initial selection of cells expressing Cse4 withΔ70 and ERS* mutations in cis (Δ70X-ERS*+Y, X +Δ70Y-ERS*), trans (X-ERS* +Δ70Y, Δ70X + Y-ERS*) or double mutant (Δ70X-ERS* +Δ70Y-ERS*) combinations. 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. (B) Growth assay. Serial dilutions of the indicated strains were plated on YPD and incubated at 30°C for 3 days. Strains analyzed were: X + Y (PKY5476), Δ70X-ERS*+Y (PKY5515), X +Δ70Y-ERS* (PKY5518), X-ERS* +Δ70Y (PKY5521) andΔ70X + Y-ERS* (PKY5524).

https://doi.org/10.7554/eLife.37911.012
Figure 4—figure supplement 3
Resequencing of plasmids.

Data from the indicated strains in shown, with the PCR-amplified product from the LEU2 plasmid on the left; the TRP1 plasmid data is on the right for those strains that had a cse4 allele on both. Residues different from wild-type Cse4 are indicated by colored boxes: X-Y interface (black), Chimera alterations (green), H3-like C-terminus (red). For the sake of space, only the 3’ 120 nt of the sequencing data is shown. No alterations from the input sequences were observed, including in the L1 and N-terminal regions that are not shown here.

https://doi.org/10.7554/eLife.37911.013

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All data generated or analysed during this study are included in the manuscript and supporting files.

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