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A two-step mechanism for epigenetic specification of centromere identity and function

Nature Cell Biology volume 15, pages 1056–1066 (2013)Cite this article

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Abstract

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

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Figure 1: CENP-A conditional disruption in human cells.
Figure 2: Disrupted centromere positioning and kinetochore nucleation requires almost complete loss of CENP-A.
Figure 3: Short-term rescue of centromere maintenance and function following CENP-A gene depletion.
Figure 4: H3CATD is sufficient for centromere identity and to template its chromatin replication.
Figure 5: N- or C-terminal tails of CENP-A nucleate kinetochore assembly.
Figure 6: The CENP-A N-terminal tail controls CENP-B levels at centromeres.
Figure 7: The fission yeast CATD is necessary and sufficient for centromere identity, but requires addition of the CENP-ACnp1 N terminus to provide long-term centromere function.
Figure 8: Model for centromeric identity, maintenance and function through a two-step mechanism.

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Acknowledgements

The authors would like to thank P. Jallepalli (Sloan-Kettering, New York, USA) for helpful suggestions, J. F. Mata and M. C. C. Silva (Gulbenkian Institute, Oeiras, Portugal), T. Panchenko (University of Pennsylvania, Philadelphia, USA) for technical help, D. Foltz (University of Virginia, Charlottesville, USA), A. Straight (Stanford University, USA), P. Maddox (Université de Montréal, Canada), S-T. Liu (University of Toledo, USE), A. Miyawaki (RIKEN, Japan), R. Allshire (WTCCB, Edinburgh, UK), Y. Watanabe (University of Tokyo, Japan) and O. Limbo and P. Russell (TSRI, La Jolla, USA) for providing reagents. We also thank B. E. Black and C. Bartocci (TSRI, La Jolla) for helpful comments on the manuscript, E. Khaliullina for drawing the model in Fig. 8, the Neuroscience Microscopy Shared Facility (P30 NS047101, University of California, San Diego) and the FACS facility in the HESCCF (Sanford Consortium for Regenerative Medicine, La Jolla, CA). This work was supported by a grant (GM 074150) from the National Institutes of Health to D.W.C., who receives salary support from the Ludwig Institute for Cancer Research. D.F. was supported by a European Molecular Biology Organization (EMBO) long-term fellowship.

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  1. H. Diego Folco & Andrew J. Holland

    Present address: Present addresses: Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA (H.D.F.); Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA (A.J.H.),

Authors and Affiliations

  1. Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA

    Daniele Fachinetti, H. Diego Folco, Yael Nechemia-Arbely, Kristen Nguyen, Alex J. Wong, Andrew J. Holland, Arshad Desai & Don W. Cleveland

  2. Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, P-2780-156, Oeiras, Portugal

    Luis P. Valente & Lars E. T. Jansen

  3. Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA

    Quan Zhu

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Contributions

H.D.F. and A.D. designed and performed yeast experiments and contributed to manuscript writing. L.P.V. and L.E.T.J. performed FISH experiments. L.E.T.J. targeted the first CENP-A allele. D.F., Y.N-A., K.N., A.J.W., Q.Z., A.J.H. performed the experiments. D.F. analysed the data. D.F. and D.W.C. conceived the experimental design and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 CENP-A conditional disruption in human cells.

(a) CENP-A genotypes validated in the indicated cell lines using PCR to distinguish normal (+), disrupted (-), and floxed (F) alleles, as diagramed to right. (b) To monitor growth rate, CENP-A+/+, CENP-AF/+ and CENP-A−/F RPE1 cells were counted every 2 days. Values represent the mean of three independent experiments, and error bars represent the SEM. (c) Immunoblot to determine CENP-A levels in CENP-A+/+, CENP-AF/+ and CENP-A−/F RPE1 cells. (d) Representative fluorescence images of centromeric CENP-A levels. Bar graphs show CENP-A protein intensity in the indicated cell lines. Bars represent the mean of three independent experiments (>30 cells per experiment). Error bars represent the SEM. ACA staining was used to identify centromeres in CENP-AF/+ and CENP-A−/F RPE1 cells. (e) Efficiency of excision of exons 3 and 4 in the overall cell population determined by Q-PCR after Ad-Cre transduction of CENP-A−/F RPE1 cells. Position of the primers is illustrated in the schematic at top. (f) PCR analysis on the DNA extracted from the survival colonies in the CENP-A−/F RPE1 cells after Ad-CRE treatment. Images show representative crystal violet-stained colonies for the indicated cell lines with or without treatment with Ad-CRE. (g) Schematic of introduction of the LoxPRFP-STOPLoxP-GFP gene by retroviral integration into CENP-A−/F cells, followed by FACS sorting to isolate only green fluorescing cells with both CENP-A alleles inactivated. (h) Graph shows the growth rate of CENP-AF/+ and CENP-A−/F with RFP-STOP-GFP construct of the experiment described in f. A parallel-untreated control is also shown. Cells were counted every 2 days. Values represent the mean of three independent experiments, and error bars represent the SEM. Scale bar = 5 μm.

Supplementary Figure 2 Centromeric proteins dissociate from centromeres with different kinetics during CENP-A depletion.

(a) Representative fluorescence images show the localization and intensity of CENP-P (purple bars). ACA staining was used to identify centromeres. Red bars show CENP-A intensity from Fig. 1e. Bars represent the mean of three independent experiments (>30 cells per experiment). Error bars represent the SEM. CENP-P was tracked by covalent labeling with rhodamine-benzyl guanine after stable expression of a gene encoding SNAP-tagged CENP-P (b) As in A, except that CENP-C and Ndc80 at day 0 and 7 are shown after Ad-Cre infection. Cells were arrested in monostarol for 12 hours. Yellow bars show Ndc80 intensity of the fluorescence images at the indicated days after Ad-Cre treatment. (c) As in a, but for CENP-A and CENP-I levels at day 0 and 9 after Ad-Cre infection. Interphase cells were quantified. Statistics source data are in Supplementary Table S2. Scale bar = 5 μm.

Supplementary Figure 3 Clonogenic and immunofluorescence localization assays determine that the CATD is sufficient for centromere identity and for templating its replication.

(a) CENP-A amino acid sequence and the domains, including the amino terminal tail (red), the αN helix (green), the CATD (light blue), and the carboxyl terminal tail (orange). (b) Representative fluorescence images of the localization of EYFP-rescue constructs of experiment outlined in Fig. 3. (c) Clongenic survival assays (as in Fig. 3d) for the CENP-A−/F cells with and without incubation in Ad-Cre and with the indicated rescue constructs. (d) Representative fluorescence images of the localization and intensity of CENP-T, CENP-A and EYFP-H3CATD at the indicated days after Ad-Cre treatment. Scale bar = 5 μm.

Supplementary Figure 4 H3CATD combined with either the amino or carboxyl tails of human CENP-A confer long term centromere identify and kinetochore function.

(a) (Top) Clonogenic survival assays for RPE1 cells with the indicated CENP-A genotypes and rescue constructs. (Bottom) Corresponding growth rates determined by counting cell numbers every 2 days for CENP-A−/F cells and CENP-A−/− cells with CENP-A, NH2H3CATD, or H3CATD−C rescue genes. Values represent the mean of three independent experiments, and error bars represent the SEM. (b) Confirmation of excision of endogenous CENP-A alleles in the indicated survival rescue construct cells. A schematic representation of the position of the primers is also shown. (c) (Top) Representative fluorescence images show the loss of endogenous CENP-A at centromeres only for cells lacking the amino-terminus of CENP-A epitopes recognized by our CENP-A antibody. ACA staining was used to identify centromeres. (Bottom) Quantitation of the fluorescence images in the CENP-A−/−+H3CATD−C and CENP-A−/−+NH2CENP-A cells. (d) Representative fluorescence images showing localization of the NH2H3CATDSNAP in CENP-A−/− cells during different phases of the cell cycle. ACA staining was used to identify centromeres and a-tubulin to identify telophase/early G1 cells. (e) Indirect immunofluorescence images of localization and intensity of EYFP-H3CATD−C at centromeres after GAPDH or HJURP siRNA, respectively. ACA staining was used to identify centromeres. (f) Quantitation of EYFP intensity at centromeres in images from (e) after GAPDH, HJURP siRNA, or no siRNA. Error bars represent the SEM of two independent experiments (>30 cells per experiment). ACA staining was used to identify centromeres. (g) (Left) CENP-C protein intensity after GAPDH or CENP-C siRNA in the indicated cell lines, respectively. Bars represent the mean of two independent experiments (>30 cells per experiment). (Right) Percentage of cells that undergo division after 72 hours of CENP-C depletion, determined by live cell microscopy. Error bars represent the SEM. Statistics source data are in Supplementary Table S2. Scale bar = 5 μm

Supplementary Figure 5 Accurate chromosome segregation with centromere-targeted histone H3 carrying either CENP-A tail domain.

(a) Representative fluorescence images of localization and intensity of centromere-bound CENP-I or Ndc80 in CENP-A−/− cell lines with the indicated rescue genes. ACA staining (red) is also shown. (b) Representative fluorescence images showing localization and intensity of centromere bound CENP-B in the indicated cell lines. (c) Representative fluorescence images showing localization and intensity of CENP-B in the indicated cell lines after 2 days of shRNA against CENP-B. (Right) Quantitation of CENP-B protein intensity of fluorescence images. (d) Time-lapse images of CENP-A−/−+ EYFPNH2H3CATD (green) RPE1 cells also stably expressing histone H2B-mRFP (red). Numbers at the top right refer to the time in minutes after nuclear envelope breakdown (NEBD). Yellow arrows show the formation of micronuclei. (e) (Left) Quantitation of misaligned or lagging chromosomes or subsequent micronuclei formation or (right) mitotic timing, determined from images in (d). Time in mitosis was defined by the period from NEBD to chromosome decondensation. Bars represent the mean of >50 cells per condition. Each individual point represents a single cell. Error bars represent the SEM. Statistics source data are in Supplementary Table S2. Scale bar = 5 μm.

Supplementary Figure 6 Protein level expression of the fission yeast rescue constructs.

Immunoblot to quantify levels of each GFP-tagged rescue variant, visualized with a GFP antibody. β-actin is used as a loading control.

Supplementary Figure 7 Uncropped image of blots from Figs 1c, 3c, 4d, 5c, 6b.

Supplementary Table 1 List of strains used in this study. Fission yeast strains used in Fig. 7 are listed. The source column indicates where the original strain comes from. The genotype of each strain is also indicated. [68Pidoux, A.L., Richardson, W. & Allshire, R.C. Sim4: a novel fission yeast kinetochore protein required for centromeric silencing and chromosome segregation. J. Cell Biol. 161, 295–307 (2003)].
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Supplementary Table 2 Statistic source data (n<5).
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A method to eliminate Cre-escaper cells.

Movie of CENP-A−/F cells expressing the LoxPRFP-STOPLoxP-GFP cassette one hour after infection with Ad-Cre. One frame captured every 5 minutes. Time at the top left shows number of minutes after the start of filming. (MOV 12652 kb)

Lowered levels of CENP-A corresponded with increased rates of chromosome segregation defects.

Movie of CENP-A−/F cells expressing Histone H2B-mRFP five days after infection with Ad-Cre. One frame captured every 3 minutes. (AVI 2196 kb)

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Fachinetti, D., Diego Folco, H., Nechemia-Arbely, Y. et al. A two-step mechanism for epigenetic specification of centromere identity and function. Nat Cell Biol 15, 1056–1066 (2013). https://doi.org/10.1038/ncb2805

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