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Mitotic chromosome assembly despite nucleosome depletion in Xenopus egg extracts

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Science  23 Jun 2017:
Vol. 356, Issue 6344, pp. 1284-1287
DOI: 10.1126/science.aam9702
  • Fig. 1 Mouse sperm nuclei can be converted into mitotic chromatids in Xenopus egg extracts.

    (A) Preparation of mouse sperm nuclei. (B) Immunofluorescence of mouse sperm nuclei incubated with a Xenopus egg extract. At the indicated time points, chromatin was labeled with anti-CAP-E, antihistone H3, and DAPI. (C) Centromeres and telomeres were labeled with anti-centromere antibody (ACA) and anti-TRF2, respectively. Presumed structure of a mouse sperm-derived chromatid is depicted in the illustration. (D) Immunoblotting of proteins that associate with mouse or Xenopus sperm nuclei incubated with egg extracts. (E) Protein composition of mitotic chromatids assembled from mouse sperm nuclei in Xenopus egg extracts. The gel was stained with Coomassie blue. Scale bars, 5 μm.

  • Fig. 2 Asf1 depletion impairs nucleosome assembly.

    (A) Putative scheme for nucleosome assembly on mouse sperm DNA. (B) Immunofluorescence of mouse sperm nuclei incubated with Δmock or ΔAsf1 extracts for 180 min. Chromatids were labeled with anti-topo II, antihistone H3, and DAPI. (C) Line profiles of normalized signal intensities of DAPI and anti-topo II. Lines drawn perpendicular to chromatid axes were analyzed. The mean and SD of 20 lines are shown. (D) Micrococcal nuclease (MNase) digestion assay of chromatids assembled in the indicated extracts. The gel was stained with ethidium bromide. (E) Protein compositions of mitotic chromatids assembled in the indicated extracts. The gel was stained with Coomassie blue. Scale bar, 5 μm.

  • Fig. 3 Nucleosome-depleted mitotic chromatids have sparse and fragile chromatin loops.

    (A) Direct comparison of clusters of chromatids assembled in Δmock and ΔAsf1 extracts. Fixed samples derived from the two different reactions were spun onto a single coverslip and processed for immunofluorescence. (Top) Distribution of the intensity of DAPI signals. (Bottom) Immunofluorescence for H3 and topo II. Nucleosome-containing and nucleosome-depleted chromatids are indicated by the arrows and arrowheads, respectively. (B) The compaction indexes (the average DAPI intensities per unit area) were plotted. (C) DNase I digestion assay. Fixed chromatids [prepared as in (A)] were treated with or without the nuclease. (D) Immunofluorescence for RPA. (E) The numbers of RPA foci per each nucleus were plotted. Bars indicate median and interquartile ranges. ***P < 0.0001, Mann-Whitney U test, (B) and (E). Scale bars, 10 μm.

  • Fig. 4 Functional cross-talk between condensins and nucleosomes is vital for full compaction of chromatids.

    (A) Blow-up images of mitotic chromatids assembled in the egg extracts indicated. The resultant chromatin was labeled with anti-CAP-G (condensin I) and anti-CAP-H2 (condensin II). (B) Line profiles of normalized signal intensities of CAP-G and CAP-H2. The mean and SD of 20 lines are shown. (C) A model for mitotic chromosome assembly proposed in the current study. Scale bar, 5 μm.

Supplementary Materials

  • Mitotic chromosome assembly despite nucleosome depletion in Xenopus egg extracts

    Keishi Shintomi, Fukashi Inoue, Hiroshi Watanabe, Keita Ohsumi, Miho Ohsugi, Tatsuya Hirano

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S8
    • Tables S1 and S2
    • References

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