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Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

Nature Cell Biology volume 16, pages 281–291 (2014)Cite this article

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Abstract

To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin–dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors such as CAF-1, DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance.

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Figure 1: Isolation of replication forks and nascent chromatin by NCC technology.
Figure 2: Genome maintenance factors at the fork.
Figure 3: Chromatin assembly and maturation.
Figure 4: Histone mark dynamics during chromatin maturation.
Figure 5: Identification of uncharacterized proteins with a predicted function in chromatin replication.
Figure 6: Localization of uncharacterized proteins with a predicted function in chromatin replication.
Figure 7: FAM111A facilitates PCNA loading and S-phase entry.
Figure 8: Chromatin dynamics during DNA replication.

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Acknowledgements

We would like to thank J. Déjardin for fruitful discussions, K. Helin for reagents (Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Denmark), Ib J. Christensen for support on statistics, and C. Wu, W. C. Earnshaw and Z. Jasencakova for critical reading of this manuscript. The A.G. laboratory is supported by a European Research Council Starting Grant (ERC2011StG, no. 281,765), the Lundbeck Foundation, the Danish Cancer Society, the Danish National Research Foundation (DNRF82) and Medical Research Council, the Novo Nordisk Foundation and FP7 Marie Curie Actions ITN Nucleosome4D. The Wellcome Trust generously supported this work through a Senior Research Fellowship to J.R. (084229), two Wellcome Trust Centre Core Grants (077707, 092076) and an instrument grant (091020). C.A. was supported by fellowships from HFSP and the Danish Medical Research Council. G.K. was supported by a FEBS Long-Term fellowship.

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  1. Patrice Menard & Jakob Mejlvang

    Present address: Present addresses: Novo Nordisk Foundation Center for Biosustainability, DK-2970 Hørsholm, Denmark (P.M.); Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway (J.M.).,

Authors and Affiliations

  1. Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark

    Constance Alabert, Sung-Bau Lee, Kyosuke Nakamura, Patrice Menard, Jakob Mejlvang & Anja Groth

  2. Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK

    Jimi-Carlo Bukowski-Wills, Georg Kustatscher, Flavia de Lima Alves & Juri Rappsilber

  3. Department of Biotechnology, Technische Universität Berlin, 13353 Berlin, Germany

    Juri Rappsilber

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Contributions

A.G., C.A. and J.R. conceived the project and designed the study. C.A., S.L., K.N. and J.M. performed and analysed experiments. J.B., G.K., F.A. and J.R. performed mass spectrometry and analysed SILAC data. P.M. generated F.A.M. mutants. The manuscript was written by C.A. and A.G. and edited by J.R., J.B. and G.K.

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Correspondence to Juri Rappsilber or Anja Groth.

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Supplementary Figure 1

(a) Biotin-dUTP labelling does not affect S phase progression. Cells synchronized in mid-S phase were labelled with biotin-dUTP during a 5 min hypotonic shift (red) or incubated in PBS (blue), and analysed by FACS at the indicated times. (b) Biotin-dUTP labelling does not trigger DNA damage as measured by γ H2AX staining. Cells were treated with hydroxyurea (HU, 3 mM) for one hour as a positive control. (c) Co-localization between PCNA and biotin-dUTP is lost during chromatin maturation. U-2-OS cells stably expressing PCNA-RFP were pulse labelled with biotin-dUTP for 20 min and fixed directly (nascent) or left for 2 h before fixation (mature). Cells not treated with biotin-dUTP are shown as a negative control. (d, e) NCC with shorter biotin-dUTP labelling times. (d) The percentage of biotin-dUTP positive cells (left) and the biotin-dUTP intensity per cell (right) after 5, 10, 20 and 40 min of labelling. Horizontal lines represent the median, ****p < 0.0001, p=0.0168, n.s. p= 0.2918 (unpaired t test, 108 < n). Statistics source data is available in Supplementary Table 4. (e) Western blot analysis of NCC pull-downs after 5 and 10 min pulse-labelling with biotin-dUTP. Labelling times of > 5 min are preferable for efficient incorporation of biotin-dUTP. The amount of starting material should always be adjusted according to labelling time in order to achieve sufficient material for a comprehensive proteomic analysis.

Supplementary Figure 2

(a) Experimental design for NCC-SILAC. Two independent cultures grown in heavy and light amino acids were released into S phase from a single thymidine block. Cells were labelled with biotin-dUTP for 20 min 3 h after release into S phase. Light cultures were left to progress in S phase for 2 h after labelling, while heavy cultures were harvested immediately. Synchronization and release of heavy cultures were shifted 2 h with respect to light cultures, such that they could be cross-linked and processed in parallel for NCC. After pull-down, the heavy and light samples were washed stringently and mixed prior to SDS-PAGE and mass spectrometry. (b) Biotin-dUTP incorporation scored by immunofluorescence verified equal labelling efficiency in heavy and light cultures. Approximately 250 cells were counted. (c) Cell cycle profile of heavy and light cultures verified matching synchronization at the time of the labelling (left) and that light cultures had progressed to late S phase at the time of harvest (right). (d) Scatter-plot comparing log2 SILAC ratios between replicates and (e) histogram of median of pairwise log2 fold difference of SILAC ratios between three replicates, illustrating reproducibility of the NCC-SILAC technology.

Supplementary Figure 3

(a) Nascent chromatin enrichment of factors proposed to deal with DNA–RNA duplexes and protein degradation at the fork20,56. (b) Nascent chromatin enrichment of origin recognition and licensing factors. (c) Coverage of the PCNA interactome by NCC-SILAC. Bars illustrate factors identified in NCC relative to those reported in the literature, numbers are indicated in brackets. Functional groups of PCNA interactors are according to Moldovan et al., 2007. For details see Supplementary Table 2. (d) Analysis of factors enriched on nascent chromatin by western blot. For comparison the log2 nascent chromatin enrichment is indicated. (e) Nascent chromatin enrichment of lysine and arginine methyltransferases (violet) and demethylases (grey). (f) Nascent chromatin enrichment of RNA polymerases. Only unique subunits are shown. All Nascent chromatin enrichments are presented as in Fig. 1h.

Supplementary Figure 4

Box plot of nascent chromatin enrichment of the individual classes defined in Figure 5a.

Supplementary Figure 5

Analysis of GFP- or FLAG-tagged proteins in U-2-OS cells stably expressing RFP-PCNA. Localization pattern (a, c) and co-localization with PCNA measured by Pearson coefficient (b, d) were scored after pre-extraction. The Pearson coefficient is calculated for individual nuclei and shown in a box plot (n ≥ 9). Horizontal line represents the median.

Supplementary Figure 6

Uncropped Western blots.

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Alabert, C., Bukowski-Wills, JC., Lee, SB. et al. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol 16, 281–291 (2014). https://doi.org/10.1038/ncb2918

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