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Reconstitution of mitotic chromatids with a minimum set of purified factors

Nature Cell Biology volume 17, pages 1014–1023 (2015)Cite this article

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Abstract

The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process.

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Figure 1: Preparation of substrate chromatin.
Figure 2: A combination of topo II and condensin I is insufficient to reconstitute mitotic chromatids from substrate chromatin.
Figure 3: A missing factor(s) essential for chromatid reconstitution is biochemically traceable.
Figure 4: A missing factor required for chromatid reconstitution is identified as the histone chaperone FACT.
Figure 5: Mitotic chromatids can be reconstituted with a minimum set of purified factors.
Figure 6: Mitotic chromatid reconstitution depends on an embryonic histone variant that is highly susceptible to FACT.
Figure 7: Cdk1-mediated phosphorylation of condensin I is sufficient to activate the reconstitution reaction.
Figure 8: A model for mitotic chromatid reconstitution.

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Acknowledgements

We thank H. Kurumizaka, B. Cairns, J. Lindsley, A. Musacchio, K. Ohsumi, M. Iwabuchi, E. Okumura, T. Kishimoto, and RIKEN Bioresource Center for reagents; R. Terui for help with antibody production; K. Otsuki, M. Usui and A. Abe for LC-MS/MS analysis; and members of the Hirano laboratory for technical assistance and critical reading of the manuscript. K.S. is particularly grateful to K. Maeshima for his insightful suggestion about PEG fractionation. This work was supported by Grant-in-Aid for Scientific Research C and Grant-in-Aid for Scientific Research on Innovative Areas (to K.S.) and Grant-in-Aid for Specially Promoted Research (to T.H.).

Author information

Authors and Affiliations

  1. Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan

    Keishi Shintomi & Tatsuya Hirano

  2. Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

    Tatsuro S. Takahashi

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  1. Keishi Shintomi
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Contributions

K.S. prepared the materials and performed the experiments; T.S.T. generated the antibodies against Spt16 and Ssrp1; K.S. and T.H. designed and analysed the experiments, and wrote the manuscript.

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Correspondence to Tatsuya Hirano.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Reconstitution of mitotic chromatids depends on titratable concentrations of condensin I and ATP.

(a) Sperm chromatin was incubated with reaction mixtures containing five factors (dX-dB, Npm, Nap1, FACT, and topo II) and decreasing concentrations of condensin I along with 1 mM ATP. After 120 min, the chromatin was fixed, labelled with anti-CAP-G antibody, and stained with DAPI. (b) Sperm chromatin was incubated with a complete mixture composed of six factors (dX-dB, Npm, Nap1, FACT, topo II, and condensin I) in the absence or presence of 1 mM ATP or ADP. After 120 min, chromatin was processed as in a. Chromatin morphology was classified as shown in Fig. 3d. Scale bars, 10 μm.

Supplementary Figure 2 Recombinant H2A-H2B dimers used in the current study.

(a) Schematic representation of the primary structures of the core histones H2A and H2B and their N-terminally deleted versions used in the current study. The boxes indicate regions that are folded into helices in the crystal structure of the nucleosome core particle. In the case of H2A.X-F2 (one of two isoforms of H2A.X-F), helical regions are predicted from its primary structure. (b) Different versions of H2A and H2B were expressed individually in bacteria and purified in combinations as indicated. The resulting H2A-H2B dimers were analysed by SDS-PAGE and stained with Coomassie Blue. (c) Sperm chromatin was incubated with the various H2A-H2B dimers or with no H2A-H2B, in the presence of a pair of histone chaperones (Npm and Nap1). After a 10-min incubation, the chromatin was treated with two different concentrations of micrococcal nuclease and electrophoresed in an agarose gel followed by ethidium bromide staining. Expected structural units of octameric and tetrameric nucleosomes are depicted in the cartoon at the bottom.

Supplementary Figure 3 Condensin I is the sole essential substrate of cyclin B-Cdk1 in the chromatid reconstitution assay.

Condensin I purified from I-HSS was first treated with cyclin B-Cdk1 and ATP for 30 min, and then mixed with the remaining five factors (HNN [Histone dX-dB, Npm, and Nap1], FACT, and topo II) and sperm chromatin. The Cdk inhibitor roscovitine was added before (0 min) or after (30 min) Cdk1-mediated phosphorylation of condensin I. After incubation for another 120 min, the resulting chromatin was labelled with DAPI and anti-CAP-G antibody. Chromatid reconstitution was inhibited when rescovitine was added at 0 min, but not at 30 min, strongly suggesting that condensin I is the sole target of Cdk1 phosphorylation. Scale bar, 10 μm.

Supplementary Figure 4 Entire scans of cropped immunoblot images.

The dashed red boxes indicate which regions of immunoblot were cropped for assembling the individual figure panels. The positions of size markers are shown in blue. Some panels include samples not mentioned in the text (indicated by the asterisks) or bands detected with antibodies unrelated to the current study (indicated by the arrows).

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Shintomi, K., Takahashi, T. & Hirano, T. Reconstitution of mitotic chromatids with a minimum set of purified factors. Nat Cell Biol 17, 1014–1023 (2015). https://doi.org/10.1038/ncb3187

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