Histone H2A is required for normal centromere function in Saccharomyces cerevisiae - PubMed (original) (raw)

Histone H2A is required for normal centromere function in Saccharomyces cerevisiae

I Pinto et al. EMBO J. 2000.

Abstract

Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G(2)-M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere-kinetochore function during chromosome segregation.

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Figures

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Fig. 1. Nucleosome model showing the position of the altered amino acids in the hta1 mutants. The positions of the single amino acid replacements in the hta1–200 and hta1–300 mutants, S20 and G30, respectively, are indicated in the model of the nucleosome based on the crystal structure generated by Luger et al. (1997). The atomic coordinates file 1aoi was obtained from Brookhaven Protein Databank (PDB) and visualized with RasMac v2.6. The DNA phosphodiester backbone is shown in white. The eight histone protein chains forming the histone octamer are shown as ribbon traces in different colors: H4, blue; H3, red; H2B, green; and H2A, yellow. Amino acids S20 (magenta) and G30 (turquoise) are evolutionarily invariant and correspond to positions S18 and G28 in the amphibian protein used for the X-ray crystallography. (A) Top view, down the DNA superhelix axis. (B) Detail from (A) showing the amino acids S20 and G30 in close contact with DNA. (C) Side view, perpendicular to the DNA superhelix axis. (D) Detail from (C), showing amino acids S20 and G30.

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Fig. 2. Flow cytometric analysis of DNA content in the hta1 mutants. (A) Comparison of exponentially growing hta1–200 (FY987), hta1–300 (FY988), hta1–950 (FY990) and hta1–1170 (FY991) mutant strains with wild-type HTA1 haploid (FY604) and HTA1/HTA1 diploid (FY604 × FY605) strains. (B) Appearance of diploids in a growing YPD culture of spores germinated from an HTA1/hta1–200 heterozygous diploid (a cross of FY605 × FY1819 followed by loss of plasmid pSAB6). The first 25 generations are present in the colony formed from the germinated spore, where most cells are arrested in G1 (1C). Flow cytometric analysis was performed on aliquots of the cultures at the indicated number of cell generations after germination.

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Fig. 3. Analysis of ploidy increase in the hta1 mutants. (A) Genetic events that result from chromosome gain in the strains marked at ade2 on chromosome XV, leading to Ade+ Leu+ papillae. Equivalent events occur on chromosome II marked at lys2. Details of the assay are explained in Materials and methods and Chan and Botstein (1993). (B) Papillation assay showing chromosome gain in the indicated strains marked at chromosomes II and XV. Chromosome V was monitored by the appearance on CanR papillae induced by UV irradiation. Strains used were: HTA1/HTA1 (FY1820), HTA1 (FY1821), hta1–200 (FY1823) and hta1–300 (FY1824).

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Fig. 4. hta1 mutants arrest at G2–M. Exponentially growing cells were synchronized in G1 with α–factor and released at 13°C. Samples were taken at the times indicated and analyzed. Strains used were: HTA1 (FY605), hta1–200 (FY1817) and hta1–300 (FY1818). (A) Cell growth in YPD. (B) DNA content by flow cytometry. (C) Quantitation of the nuclear and bud morphology. UB, unbudded; SB, small budded; LB, large budded; DB, double budded. The same cells were used for flow cytometric analysis in (B).

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Fig. 5. Microtubule and spindle pole body analysis in synchronized cells shifted to 13°C. (A) Microtubule staining by indirect immuno- fluorescence with anti-tubulin antibodies of cells taken 24 h after the shift to 13°C. Nuclear DNA was stained with DAPI. The hta1 mutant shows large-budded cells with an undivided nucleus and a short spindle. The arrowhead indicates a wild-type cell in anaphase with an elongated spindle. Arrows indicate hta1 large-budded cells with a short spindle. Strains used were: HTA1 (FY605), hta1–200 (FY1817) and hta1–300 (FY1818). (B) Spindle pole body staining by green fluorescence on live cells marked with NUF2::GFP. Cells were synchronized with α–factor and released at 13°C; a sample was taken 24 h after the shift to 13°C and analyzed by fluorescence microscopy. In the wild-type cells a single dot corresponding to an undivided spindle pole body is present in an unbudded cell (arrowhead), or two dots at the opposite poles of a large-budded cell (arrow) are present in a cell at anaphase. The two dots shown in mutants correspond to the spindle pole body that has duplicated and separated a short distance within a large-budded cell, consistent with a large-budded cell with an undivided nucleus. In some cases only one dot appears in the focal plane of the picture. (C) Quantitation of the GFP signal in the hta1–200 and hta1–300 strains. Numbers represent percentage values. Strains used were: HTA1 (FY1825), hta1–200 (FY1826) and hta1–300 (FY1827).

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Fig. 6. Sister chromatid separation in the hta1–200 mutant. Newly germinated wild-type HTA1 and hta1–200 cells were scored for sister chromatid separation and DNA content during a single cell cycle. (A) Phenotypes of HTA1 and hta1–200 cells released from α–factor arrest at 30°C (t = 0) and observed after one generation time (1.5 and 2.5 h, respectively) and 1.7 generation times (2.5 and 4.0 h, respectively). These strains carry lactose operators integrated near the centromere of chromosome III. CEN3 was visualized by fluorescence microscopy after induction of GFP–lacI. At the beginning of the cycle (t = 0) the unbudded cells contain one GFP dot representing the unreplicated chromosomes. By the end of the cycle HTA1 cells have separated their sister chromatids to opposite poles (t = 1.5), while hta1–200 cells contain cells where both sisters separate at one pole (t = 2.5, arrows). After completion of cytokinesis HTA1 cells contain single dots (t = 2.5), while hta1–200 cells contain both sisters (t = 4.0, arrowheads). (B) Single cells containing one, two or four GFP dots were scored at the beginning and end of the cycle. At least 200 cells were scored for each time point. Cells with no GFP signal (aploids) were not scored. Numbers represent percentage values of total cells with GFP signal. (C) DNA content of the samples used in (A) and (B) was analyzed by flow cytometry.

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Fig. 7. Double-mutant phenotypes caused by hta1 mutants in combination with the kinetochore mutants indicated. The genotypes correspond to the following strains: HTA1 (FY605), hta1–200 (FY1817), hta1–300 (FY1818), hta1–1170 (L992), mif2-3 (L986), hta1–200 mif2-3 (L987), hta1–300 mif2-3 (L988), cse4-1 (L989), hta1–200 cse4-1 (L990), hta1–300 cse4-1 (L991), ctf13-30 (L980), hta1–200 ctf13-30 (L981), hta1–300 ctf13-30 (L982), ctf14-42 (L983), hta1–200 ctf14-42 (L984), hta1–300 ctf14-42 (L985), hta1–1170 ctf13-30 (L993) and hta1–1170 ctf14-42 (L994). Strains were grown on YPD for 2 days at 30°C (A, B and C), for 4 days at 23°C (D) or for 4 days at 26°C (E and F).

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Fig. 8. The hta1–200 and hta1–300 mutants have altered chromatin structure over the CEN3 region. Nuclei were isolated from wild-type haploid, diploid and hta1 mutant strains after growth at permissive (30°C) or restrictive (13°C for 24 h) conditions, digested with increasing concentrations of MNase and subjected to indirect end-labeling analysis as described in Materials and methods. (A and C) _Bam_HI-digested DNA hybridized with a radiolabeled 616 bp DNA fragment adjacent to the restriction site. (B) _Bst_BI-digested DNA hybridized with a radiolabeled 173 bp DNA fragment adjacent to the restriction site. Positions of the CEN3 nuclease-resistant core and the flanking nucleosomes altered in the mutant are indicated in the diagram to the right. Positions of the probes are indicated as open bars. Arrows and bullets represent enhanced and diminished MNase digestion in the hta1–200 and hta1–300 mutants, respectively, compared with wild type. Strains are as follows: N, naked DNA from FY604, HTA1 (FY604), HTA1/HTA1 (FY604/FY605), hta1–200 (FY987), hta1–300 (FY988) and hta1–950 (FY990). Strains FY987 and FY988 are homogeneous diploid populations. (D) Summary of the MNase digestion pattern shown by the hta1–200 and hta1–300 mutants at the CEN3 locus. Dashed arrows represent diminished digestion and thicker arrows represent enhanced digestion. The eight nucleosomes altered in the hta1 mutant are depicted by dashed ovals.

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Fig. 9. High-copy-number suppression of the cold sensitivity and increase in ploidy of the hta1 mutants. Strains containing the 2 μm vector pRS425 or ACT3 carried on the same vector were grown as patches on 5–FOA-Leu to allow the hta1 mutant strains to lose the episomal wild-type HTA1 gene carried on a _URA3_-based plasmid, and then replica printed on the media indicated. Cold sensitivity was monitored by growth on YPD plates for 2 weeks at 13°C, increase in ploidy was monitored by the appearance on CanR papillae induced by UV irradiation after 4 days at 30°C, SUC2 expression was monitored by growth on raffinose plates grown at 30°C for 2 days. Strains used were: HTA1/HTA1 (FY604 × FY605), HTA1 (FY604), hta1–200 (FY1819) and hta1–300 (FY1897).

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