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Epigenetically induced paucity of histone H2A.Z stabilizes fission-yeast ectopic centromeres

Nature Structural & Molecular Biology volume 20, pages 1397–1406 (2013)Cite this article

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Abstract

In most eukaryotes, centromeres are epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A–loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at noncentromeric regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe that show reduced association with the CENP-A chaperone Scm3, owing to persistence of the histone H2A variant H2A.Z. After the acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, thus leading to subsequent stabilization of the neocentromeres. These findings provide new insights into histone variant–mediated epigenetic control of neocentromere establishment.

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Figure 1: Identification and characterization of Δcen3-NCs.
Figure 2: Reduced association of Cnp1 and Scm3 with Δcen3-NCs.
Figure 3: Defective chromosome III segregation in Δcen3-NC survivors.
Figure 4: Emergence of type-F and type-R revertants from Δcen3-NC cells.
Figure 5: Pht1 depletion promotes Cnp1 enrichment through efficient Scm3 recruitment.
Figure 6: Loss of rDNA repeats permits efficient NC formation on chromosome III.
Figure 7: The contribution of heterochromatin to Δcen3-NC maturation.
Figure 8: Schematic model summarizing ectopic centromere establishment in S. pombe.

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Acknowledgements

We thank Y. Hiraoka (Osaka University) for the GFP visualization plasmids, H. Matsuzaki (Fukuyama University) for the R-recombinase clone and K. Gull (University of Oxford) for the TAT1 antibodies. We also thank H. Kimura and T. Stasevich (Osaka University) for critical reading of the manuscript. This study was supported by Grants-in-Aid for Young Scientists (A) (K.I., 21687014), Scientific Research (B) (K.I., 24370003) and Challenging Exploratory Research (K.I., 22657002 and 25650122) from the Japan Society for the Promotion of Science (JSPS), Grant-in-Aid for Scientific Research on Innovative Areas (Genome Adaptation) (K.I., 22125004) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Osaka University Life Science Young Independent Researcher Support Program (K.I.) and the Naito Foundation (K.I.). Y. Ogiyama and Y. Ohno are supported as JSPS Fellows.

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  1. Laboratory of Chromosome Function and Regulation, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

    Yuki Ogiyama, Yuko Ohno, Yoshino Kubota & Kojiro Ishii

  2. Institute for Academic Initiatives, Osaka University, Osaka, Japan

    Kojiro Ishii

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  1. Yuki Ogiyama
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  2. Yuko Ohno
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Contributions

Y. Ogiyama and K.I. designed the experiments. Y. Ogiyama, Y.K. and K.I. performed the experiments. Y. Ogiyama and K.I. analyzed the data. Y. Ohno performed the sequencing analysis of NC-accommodating genomes. Y. Ogiyama and K.I. interpreted the data, and K.I. wrote the paper.

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Correspondence to Kojiro Ishii.

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Integrated supplementary information

Supplementary Figure 1 Analysis of Cnp1 association with NCs.

(a,b) The chromosomal distribution of Cnp1 (red) and subtelomeric heterochromatin (dimethylated histone H3 K9 (H3K9me2); blue) along the Δcen1-NCs (left subtelomeric region (tel1-L, a) and right subtelomeric region (tel1-R, b)) in the indicated strains. A repeat-suppressed S. pombe genomic microarray was used for the ChIP-chip analysis. The repetitive regions suppressed in the microarray are indicated in yellow. The x-axis numbers represent the chromosome I coordinates. The NCs in cd1-46 and cd1-50 were previously mapped to the same loci as those in cd1-39 and cd1-60, respectively8, and exhibit similar Cnp1 distributions. The variation of Cnp1 distribution along the NCs infrequently formed in the absence of heterochromatin (Δclr4 cd1-132 and Δclr4 cd1-136)8 was within the range of that seen in the wild-type. For reference, the positions of the quantitative chromosome I PCR probes (a–e) used elsewhere in this study are also indicated. (c,d) The chromosomal distribution of Cnp1 along the Δcen2-NCs (cd2-163 (c) and cd2-166 (d)). A repeat covering S. pombe genomic microarray was used for the ChIP-chip analyses. The corresponding chromosome II regions (tel2-L (c) and tel2-R (d)) are displayed in alignment with the Sanger Center S. pombe genome coordinates. Annotated genes are indicated by arrows or arrowheads and essential genes are displayed in pink. (e) NC mapping of 20 Δcen2-NC survivors showing Cnp1 ChIP enrichments in each strain. All of the Δcen2-NCs were mapped to either tel2-L or tel2-R and none were mapped to the mat locus. (f,g) The chromosomal distribution of Cnp1 along the NCs obtained in the Δcen3 experiments (cd3-389 (f) and cd3-385 (g)). The ChIP-chip analysis and data presentation were performed as described in c,d. Only four Δcen3-NC survivors were obtained in total, and the ChIP-chip results for the two other survivors (Y. Ogiyama and K.I., unpublished data) were almost identical to those for cd3-389. For reference, the positions of the chromosome III quantitative PCR probes (a–j) used elsewhere in this study are also indicated.

Supplementary Figure 2 Irregular chromosome III segregation in Δcen3-NC cells.

(a) Quantitative ChIP enrichment of Cnp1 protein across the Δcen3-NCssp2 region in cd3-385 and the Δcen3-NCrDNA region in cd3-389. The positions of the quantitative PCR probes (a–j) in chromosomes III are shown in Supplementary Figure 1. The ChIP enrichments were normalized to those of the cnt2 region in cen2 (cen2). The data are represented as the average + s.e.m. of n = 3 biological repeats. (b) A typical fluorescent microscopy image of GFP-Cnp1-visualized centromeres (green), Nuc1-mCherry-visualized rDNAs50 (magenta), and Hoechst 33342-stained DNAs (blue) of Δcen3-NC cells (cd3-385) harboring the nda3-KM311 cold sensitive mutation35. The cells were arrested in mitosis by incubation at 18°C for 10 h. Cells exhibiting more than two GFP-Cnp1 dots connected to an rDNA signal, in addition to two other GFP-Cnp1 dots corresponding to canonical cen1 and cen2, are indicated by arrowheads. The cell that had lost chromosome III (no rDNA signal) is indicated by an arrow. Scale bar, 10 μm.(c) The percentages of cells exhibiting loss or gain of chromosome III. The indicated strains were mitotically arrested as described in b. The rDNA signals tended to be clustered and were difficult to distinguish even in the nda3-arrested nucleus; therefore, the numbers of GFP-Cnp1 dots associated with the rDNA-containing chromosome in each nucleus were counted. Increases in irregular numbers of rDNA-connected GFP-Cnp1 dots (colored bars) in Δcen3-NCrDNA cells (cd3-389) and Δcen3-NCssp2 cells (cd3-385) are indicative of chromosome III mis-segregation. 7.4% of the Δcen3-NCssp2 cells harbored an rDNA-containing chromosome III without any GFP-Cnp1 dots (cyan), suggesting a loss of NC from the chromosome. Note that the quantification of GFP-Cnp1 signal intensities shown in Figure 2d was performed using only the cells exhibiting a regular single GFP-Cnp1 dot connected to an rDNA signal (gray).

Supplementary Figure 3 Profiles of the centromere deletion screens and resultant survivor characterizations.

(a) The viabilities of total cells (blue) and centromere-deleted cells (magenta), as determined by their resistance to G418 and 5-fluoroorotic acid (see METHODS). Error bars represents s.e.m. of n = 6 biological replicates. loxP-cen1, loxP-cen2, and loxP-cen3, the strains used for deleting cen1, cen2, and cen3, respectively. (b) The relative ratios between NC formation and telomere fusion in the survivors obtained in each screen. (c) Cell growth of the wild-type, NC survivors, and revertant cells growing on YES media at 33°C. The generation times of each strain were as follows: wild-type, 2.1 h; cd1-39, 1.9 h; cd1-60, 2.0 h; cd2-163, 2.1 h; cd2-166, 2.2 h; cd3-385, 2.7 h; cd3-386, 3.0 h; and cd3-386-r1, 2.0 h. (d) A typical fluorescent microscopic image of Hoechst 33342-stained anaphase chromosomes (magenta). Chromosome III visualization was obtained by tandem tethering of the GFP-LacI protein (green). Chromosome III was segregated equally to the daughter nuclei in wild-type cells but not Δcen3-NC cells (cd3-385). Scale bar, 10 μm. (e) The frequency of chromosome I segregation failure in the defective cells displaying lagging chromosomes of the indicated strains (n > 50). Cells were cultured at 33°C. One locus in chromosome I was visualized by tandem tethering of the GFP-LacI protein. Error bars represents s.e.m. of triplicate cultures. One third of the lagging chromosomes observed in heterochromatin-abrogated Δclr4 cells were derived from chromosome I, irrespective of whether it harbored canonical cen1 or Δcen1-NC, suggesting that not every NC results in lagging chromosomes.

Supplementary Figure 4 The behavior of spontaneous revertants of Δcen3-NCs.

(a) A color image of a typical colony showing the emergence of spontaneous revertants from the Δcen3-NC strain. Δcen3-NC cells (cd3-386) were grown continuously for 30 generations and were allowed to form colonies on YES plates containing phloxine B (PhB). (b) The proportions of revertants that emerged from the indicated Δcen3-NC strains during continuous cell culture. (c) PFGE separation of the chromosomes in the type-F revertants showing the reduction in chromosome number. m, molecular size marker. (d) SfiI-digested chromosomes of the type-F revertants separated by PFGE and stained with ethidium bromide (EtBr) or subjected to Southern blot (SB) analysis with the chr3L, chr3R, tel1L, and tel2R probes to observe telomere-to-telomere fusion. (e) PFGE separation of the chromosomes in the type-R revertants showing the three-chromosome configurations. m, molecular size marker. (f) The percentages of normal (gray) and defective (magenta) chromosome segregation patterns in anaphase cells. The percentages were determined as described in Figure 3e (n > 100). Error bars represents s.e.m. of triplicate cultures. Note that segregation failure occurs less frequently in the type-R revertants than in the heterochromatin-deficient Δswi6 cells and Δclr4 cells (see Fig. 3e). Furthermore, the Δswi6 mutation in the type-R revertant conferred a low level of lagging chromosomes (cd3-389-r1 Δswi6). This level was similar to that observed in the Δswi6 mutant, but different from that observed in the original Δcen3-NCrDNA cells (cd3-389). (g) SfiI-digested chromosomes of the type-R revertants separated by PFGE and stained with EtBr or subjected to Southern blot analysis with the indicated probes to show the reduction in the number of rDNA repeats in the left arm. (h) PstI-digested chromosomes of the type-R revertants separated by PFGE and subjected to Southern blot analysis with the rDNA probe. Given that there is no PstI restriction site in the 10.8-kb rDNA repeat units and the non-rDNA repeat region of the left-most PstI fragment in chromosome III encompasses approximately 3.4 kb (chrIII, 24571–28056), the rDNA 15-kb PstI fragment in cd3-386-r1 and cd3-390-r1 that hybridized to the probe most likely harbors only one copy of the rDNA repeat, and the 25-kb PstI fragment in cd3-389-r1 most likely harbors two copies.

Supplementary Figure 5 Associations of Cnp1, Scm3, Pht1 and heterochromatin with Δcen3-NCs and derivatives.

(a,e) Quantification of Cnp1 association with Δcen3-NCrDNA (a) and Δcen3-NCssp2 (e). The ChIP enrichments were normalized to the cnt2 region in cen2 (cen2). The probe positions are indicated in Supplementary Figure 1. The data are represented as the average + s.e.m. of n = 3 biological repeats. (b,c,f,g) Changes in the association of Scm3-GFP (b,f) and Pht1-GFP (c,g) with Δcen3-NCs following the heterochromatin supplementation. The ChIP enrichments across the Δcen3-NCrDNA (b,c) and Δcen3-NCssp2 (f,g) regions were quantified and normalized to those of the cnt2 region in cen2 (cen2) (b,f), and the Pht1-enriched region (SPAC1486.08)40 (c,g), respectively. The probe positions (a–j) in chromosome III are shown in Supplementary Figure 1. Data are represented as the average + s.e.m. of n = 3 biological replicates. Like the type-R revertant (cd3-389-r1), the addition of heterochromatin to the Δcen3-NCrDNA region (cd3-389+dh2.1) caused Pht1 depletion, and the Δswi6 mutation37 reversed the Pht1 depletion effect (cd3-389+dh2.1 Δswi6) (c). A similar effect was observed, albeit to a lesser extent, at the Δcen3-NCssp2 region (g). The changes in Scm3 association with Δcen3-NCs were inversely proportional to the changes in Pht1 association (b,f). However, Scm3 accumulation in the type-R revertant was not reversed by the Δswi6 mutation (cd3-389-r1 Δswi6) (b). (d,h) Quantification of artificial heterochromatin around Δcen3-NCrDNA (d) and Δcen3-NCssp2 (h). The ChIP enrichments were normalized to the heterochromatin at the canonical centromeres (cen-otr). The probe positions are indicated in Supplementary Figures 8c,d. Artificial heterochromatin formation was more prominent at the Δcen3-NCrDNA region than the Δcen3-NCssp2 region, which paralleled the replenishment of Cnp1 at the Δcen3-NCs (a,e). The data are represented as the average + s.e.m. of n = 3 biological repeats.

Supplementary Figure 6 The inverse relationship between Cnp1 and Pht1 association with NCs.

(a,b,f,g) Chromosomal distributions of Pht1-GFP (light green) and Cnp1 (magenta) in Δcen1-NC cells (cd1-39 (a) and cd1-60 (b)) and Δcen3-NC cells (cd3-389 (f) and cd3-385 (g)). Normalization and smoothing of ChIP-chip data were performed as described in Figure 5a. The 120-kb segments used for a higher magnification shown in Figure 5b ((i) to (iv)) are also indicated by blue lines. The apparent Cnp1 enrichments at the right terminus of chromosome III in cd3-389 (f) most likely result from false mapping of Cnp1 signals at the left terminus, because the right signals corresponded only to genomic regions that are conserved between both termini. (c) A higher magnification of the Pht1 distributions across the 120-kb segments encompassing canonical centromeres and NC-competent subtelomeric regions in wild-type (light green) and Δmsc1 cells (dark green). ChIP-chip data were processed as described for (a,b,f,g), except that an 11-probe window was used instead of a 201-probe window for the smoothing. The positions corresponding to Cnp1-containing centric chromatin and heterochromatin are indicated by filled rectangles. (d) Quantification of GFP-Cnp1 signals in wild-type and Δmsc1 cells in interphase. The analysis was performed as described in Figure 5c. a.u., arbitrary units. (e) Expression of the GFP-tagged scm3+ gene in wild-type, Δmsc1, and Δswi6 cells, as determined by quantitative RT-PCR. Data were normalized to act1+ expression levels and are represented as the average ± s.d. of n = 3 technical repeats.

Supplementary Figure 7 Centromere deletion screens of the t(1Rter;3Lter) and Δmsc1 strains.

(a) The viabilities of total cells (blue) and centromere-deleted cells (magenta) in the course of centromere deletion screens (loxP-cen1 and loxP-cen3) of the indicated strains. Error bars represents s.e.m. of n = 6 biological replicates. (b) The ratios of NC formation to telomere fusion in the survivors obtained in each screen. (c) NC mapping of six Δcen3-NC survivors obtained from the t(1Rter;3Lter) loxP-cen3 strain showing the Cnp1 ChIP enrichment values for each strain. All of the Δcen3-NCs were mapped to the region corresponding to Δcen3-NCrDNA (rDNA(L)). (d) NC mapping of six Δcen1-NC survivors obtained from the t(1Rter;3Lter) loxP-cen1 strain. All of the Δcen1-NCs were mapped to the left subtelomeric region of chromosome I (tel1-L) and none were mapped to the right subtelomeric region (tel1-R), even though NCs are formed more preferentially at tel1-R than tel1-L in wild-type cells8.

Supplementary Figure 8 The contribution of heterochromatin to Δcen3-NC maturation.

(a) A ChIP-chip analysis showing the acquisition of robust heterochromatin (H3K9me2; blue) near Δcen3-NCrDNA (Cnp1; red) in the type-R revertant (cd-389-r1, top), but not in the original Δcen3-NC cells (cd3-389, bottom). The yellow arrows indicate rDNA repeats. (b) Verification of the size of the rDNA repeats by PFGE of SfiI-digested chromosomes and subsequent Southern blot analysis (SB) with the indicated probes. Both the left and right termini of chromosome III (3L and 3R, respectively) showed similar size variations among the mock and dh2.1 integrants, indicating no marked reductions in the number of rDNA repeats. (c,d) ChIP-chip analysis confirming the accumulation of dimethylated histone H3 K9 modification (H3K9me2; blue) at the heterochromatin artificially formed next to the Cnp1-assembled NCs (red) in the Δcen3-NCrDNA strain (cd3-389) (c) and the Δcen3-NCssp2 strain (cd3-385) (d) following insertion of the dh2.1 DNA fragment (vertical arrow, top panels), but not following a mock insertion (bottom panels). The blue-shaded area indicates the integrated plasmid DNA region and the yellow arrows indicate rDNA repeats.

Supplementary Figure 9 Original images of gels and blots corresponding to Figure 4c,e and a validation of antibodies.

(a) PFGE analysis. m, molecular size marker. (b) Southern blot analysis with the rDNA probe. (c) Western blot analysis of S. pombe whole cell lysates with anti-Swi6 antibodies.

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Ogiyama, Y., Ohno, Y., Kubota, Y. et al. Epigenetically induced paucity of histone H2A.Z stabilizes fission-yeast ectopic centromeres. Nat Struct Mol Biol 20, 1397–1406 (2013). https://doi.org/10.1038/nsmb.2697

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