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Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion

Nature Structural & Molecular Biology volume 21, pages 617–625 (2014)Cite this article

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Abstract

Nucleosomes are the fundamental unit of chromatin, but analysis of transcription-independent nucleosome functions has been complicated by the gene-expression changes resulting from histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation and by analyzing the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that although nucleosome-free DNA can recruit nuclear-envelope membranes, nucleosomes are required for spindle assembly and for formation of the lamina and of nuclear pore complexes (NPCs). We show that, in addition to the Ran G-nucleotide exchange factor RCC1, ELYS, the initiator of NPC formation, fails to associate with naked DNA but directly binds histone H2A–H2B dimers and nucleosomes. Tethering ELYS and RCC1 to DNA bypasses the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.

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Figure 1: Histone depletion and analysis of mitotic nucleosome functions.
Figure 2: The RCC1- and Aurora B–dependent spindle-assembly pathways both require nucleosomes.
Figure 3: Proteomic profiling of nucleosome beads and DNA beads.
Figure 4: NPC assembly, but not membrane recruitment, requires nucleosomes.
Figure 5: Nucleosomes directly recruit ELYS.
Figure 6: The C-terminal domain of ELYS interacts directly with histone H2A–H2B.
Figure 7: Targeting RCC1 and ELYS to DNA is sufficient for NPC assembly in the absence of nucleosomes.
Figure 8: Roles of nucleosomes in spindle assembly and nuclear-envelope assembly.

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Acknowledgements

We thank C.D. Allis (Rockefeller University), G. Almouzni (Institut Curie), Y. Azuma (University of Kansas), R. Heald (University of California, Berkeley), M. Hetzer (Salk Institute), T. Hirano (RIKEN), I. Mattaj (European Molecular Biology Laboratory), T. Muir (Princeton University), N. Nozaki (MAB Institute, Inc.), J. Ravetch (Rockefeller University), D. Shechter (Albert Einstein University), D. Shumaker (Northwestern University), A. Straight (Stanford University) and J. Walter (Harvard Medical School) for reagents; the Rockefeller University Bio-Imaging Resource Center and D. Wynne for help with image analysis; H. Molina and J. Fernandez at the Proteomics Resource Center for MS analysis; T. de Lange, S. Giunta, K. Ura, D. Wynne, and J. Xue for critical reading of the manuscript; and members of the Funabiki laboratory for discussions. This study was supported by Austrian Science Fund grant J-2918 (C.Z.), a Marie-Josée and Henry Kravis fellowship (C.Z.), grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the New Energy and Industrial Technology Development Organization of Japan (H.K.) and US National Institutes of Health grant R01-GM075249 (H.F.).

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Authors and Affiliations

  1. Laboratory of Chromosome and Cell Biology, Rockefeller University, New York, New York, USA

    Christian Zierhut, Christopher Jenness & Hironori Funabiki

  2. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan

    Hiroshi Kimura

  3. Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Suita, Japan

    Hiroshi Kimura

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  1. Christian Zierhut
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  2. Christopher Jenness
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Contributions

C.Z. designed the study, carried out experiments and analyzed the data. C.J. carried out experiments and analyzed data. H.K. generated antibodies. H.F. designed and supervised the study. C.Z., C.J. and H.F. wrote the manuscript with contributions by H.K.

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Correspondence to Christian Zierhut or Hironori Funabiki.

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Integrated supplementary information

Supplementary Figure 1 Description of ΔH3–H4 extracts.

(a) Western blot analysis (top) and quantification (bottom) of H3 in extract compared to recombinant standards. The vertical line indicates irrelevant lanes that were removed in Adobe Photoshop. (b) Western blot analysis of the comparison of various antibodies for H3–H4 depletion. The indicated antibodies were used for H3–H4 depletion and supernatants and bead fractions were analyzed by western blot. H3K4unmod, raised against the unmodified H3 tail. H4N, antibody raised against the N terminus of H4. H3C, antibody raised against the C terminus of H3. m IgG, control mouse IgG1 antibodies. Vertical lines indicate that irrelevant lanes on the membranes were removed in Adobe Photoshop. (c) Agarose gel electrophoresis analysis of plasmids purified from E. coli or from the indicated extracts. The gel was run in the absence of intercalators. (d) Agarose gel analysis of nucleosome formation and spacing. DNA was incubated in the indicated extracts and subjected to micrococcal nuclease (MNase) digestion.

Supplementary Figure 2 Agarose gel analysis of beads recovered from extract and analysis of CPC interaction with mutant H3 N-terminal peptides.

(a) Agarose gel analysis of DNA extracted from nucleosome beads or DNA beads recovered from ΔH3–H4 extract. (b) Western blot analysis of CPC binding to the indicated peptides. H3_scr, scrambled H3 tail. H3_S3ph, Thr3 was changed to phosphorylated serine.

Supplementary Figure 3 Analysis of the reproducibility of the MS dataset.

(a) The ratio of protein quantity co-purified with nucleosome-beads over DNA-beads with ≥3 peptide spectral matches in two independent experiments was determined for each replicate. Proteins in the upper right quadrant were reproducibly enriched on nucleosomes, while proteins in the bottom left quadrant were reproducibly enriched on DNA. Note that proteins exclusively identified on DNA or nucleosomes are not plotted. (b,c) Abundance (LC-MS/MS integration) of nucleosome (b) and DNA (c) associated proteins identified with ≥3 peptide spectral matches in two independent experiments was calculated for each experiment.

Supplementary Figure 4 Characterization of proteins identified in LC-MS/MS.

(a-d) Western blot analysis of proteins co-purified with DNA beads or nucleosome beads incubated in ΔH3–H4 extracts. Each panel represents a separate experiment. (e) Coomassie staining of uncoated beads, naked DNA beads or nucleosome beads incubated with recombinant Topo II and BSA in buffer.

Supplementary Figure 5 Analysis of nuclear envelopes in ΔH3–H4 extract.

(a) Fluorescence microscopy analysis of nucleosome beads or DNA beads recovered from interphase ΔH3–H4 extracts and stained with anti-GFP antibody (green) and DAPI (magenta). Sum intensity projections of deconvolved Z stacks are shown. Scale bar, 3 μm. (b) Fluorescence microscopy analysis and quantification (c) of nucleosome beads or DNA beads recovered from interphase ΔH3–H4 extracts and stained with anti-Nup96 antibody (green) and DAPI (magenta). Single deconvolved z sections are shown. Scale bar, 3 μm. Each data point is the average intensity per bead of an individual cluster of beads (n = 15 for nucleosome beads; n = 14 for DNA beads). Median values and interquartile ranges are indicated. P < 0.0001 (two-tailed Mann-Whitney U test). (d) Western blot analysis of pull downs of nucleosome beads, DNA beads or uncoupled beads recovered from interphase ΔH3–H4 extracts. (e) Fluorescence microscopy analysis of nucleosome beads recovered from interphase or M phase ΔH3–H4 extracts and stained with anti-ELYS antibody (green) and DAPI (magenta). Scale bar, 3 μm.

Supplementary Figure 6 Analysis of nucleosome binding of ELYS and ELYSΔC fragments.

(a) Coomassie stained gel depicting the recombinant ELYS fragments used in this study, depicted in Fig. 5d. (b) Coomassie stained gel of pull downs of nucleosome beads or uncoupled beads incubated with the indicated recombinant ELYS fragments. ELYSC 2A contains two alanine mutations at R2332 and R2334 within the AT-hook. A weakly stained version and a stronger stained version of the same gel are shown. (c) Western blot analysis of pull downs of nucleosome beads or uncoupled beads incubated with the indicated recombinant ELYS fragments. Coomassie staining could not be used because ELYSC3 runs at about the same size as histones. (d) Western blot analysis of pull downs of nucleosome beads or uncoupled beads incubated with the indicated recombinant ELYS fragments. Coomassie staining could not be used because ELYSC2 runs at about the same size as histones.

Supplementary Figure 7 Expression of RCC1-DBD and ELYSΔC2-DBD in extract.

(a) Depiction of the constructs used. ELYSΔC2-DBD is ELYS amino acids 1-2280 fused to the DNA binding domain of Xkid (DBD) followed by an HA tag. RCC1-DBD is the full length Xenopus laevis RCC1 fused to the same Xkid DNA binding domain on its C terminus. (b) Western blot analysis of expression of ELYSΔC2-DBD and RCC1-DBD in extract. The same membranes were stained with anti-HA (mouse) and anti-ELYS or anti-RCC1 (both rabbit). Secondary antibodies containing different fluorophors were used. Arrowheads point ELYSDC2-DBD in the upper overlay image.

Supplementary Figure 8 Complete scans of the gels and western blots shown in the main figures.

Dotted boxes indicate the parts that are shown in the figures. Please note that some membranes were cut into multiple strips prior to antibody detection to enable visualization of multiple antigens.

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Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1248 kb)

Supplementary Data Set 1

Proteomic analysis of nucleosome- and DNA-interacting proteins (XLSX 170 kb)

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Zierhut, C., Jenness, C., Kimura, H. et al. Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. Nat Struct Mol Biol 21, 617–625 (2014). https://doi.org/10.1038/nsmb.2845

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