Abstract

Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2,3,4,5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)—the obligate BRCA1 binding partner3—as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1–BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.

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Code availability

The code used to analyse the mass spectrometry data is publicly available at GitHub (https://github.com/lukauskas/publications-nakamura-2018-snap-h4k20me2).

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009281, and are presented in Supplementary Table 1. Unprocessed images of all gels and blots (Figs. 1c,d and 5a,c and Supplementary Fig. 2h) are provided in Supplementary Fig. 5. Source data for all graphs are provided in Supplementary Table 2. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank J. Lukas for commenting on the manuscript, researchers at the Groth laboratory for fruitful discussions, R. Baer for sharing the BARD1 plasmid and BARD1-null mouse mammary carcinoma cells, M. Kanemaki for the OsTIR1 antibody, Y. Antoku for assistance with microscopy, and J. V. Johansen for help with statistical analysis. J.R.B. is funded by a Cancer Research UK Career Development Grant (C52690/A19270). Funding for T.B. was provided by the Medical Research Council (grant number MC_UP_1102/2) and European Research Council (ERC StG number 309952). S.L. was supported by a stipend from the Biotechnology and Biological Sciences Research Council. The Groth laboratory is supported by the Danish Cancer Society, Novo Nordisk Foundation, Lundbeck Foundation, European Research Council (ERC CoG number 724436), Independent Research Fund Denmark and Neye Foundation.

Author information

Author notes

    • Giulia Saredi

    Present address: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, Sir James Black Centre, University of Dundee, Dundee, UK

    • Nhuong V. Nguyen

    Present address: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    • Peter A. Faull

    Present address: Francis Crick Institute, London, UK

  1. These authors contributed equally: Kyosuke Nakamura, Giulia Saredi, Jordan R. Becker.

Affiliations

  1. Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Kyosuke Nakamura
    • , Giulia Saredi
    • , Tracey E. Beyer
    • , Laura C. Cesa
    •  & Anja Groth
  2. Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Kyosuke Nakamura
    • , Tracey E. Beyer
    • , Laura C. Cesa
    •  & Anja Groth
  3. Wellcome Centre For Human Genetics, Oxford, UK

    • Jordan R. Becker
    •  & J. Ross Chapman
  4. Institute of Functional Epigenetics, Helmholtz Zentrum München, Neuherberg, Germany

    • Benjamin M. Foster
    • , Saulius Lukauskas
    •  & Till Bartke
  5. MRC London Institute of Medical Sciences, London, UK

    • Benjamin M. Foster
    • , Nhuong V. Nguyen
    • , Peter A. Faull
    • , Saulius Lukauskas
    •  & Till Bartke
  6. Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK

    • Benjamin M. Foster
    • , Nhuong V. Nguyen
    •  & Till Bartke
  7. Department of Chemical Engineering, Imperial College London, London, UK

    • Saulius Lukauskas
  8. Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Thomas Frimurer

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Contributions

K.N., G.S. and A.G. conceived the study. K.N. carried out the functional analysis. G.S. performed pull-downs with endogenous BARD1 and SLF1, and established tools and reagents. J.R.B. generated and carried out experiments with BARD1AID/AID cells. B.M.F. prepared materials for nucleosome pull-downs. N.V.N. prepared materials, carried out SILAC nucleosome pull-down experiments and analysed the data. T.E.B. and L.C.C. performed pull-downs with BARD1 and SLF1 mutants. P.A.F. carried out mass spectrometry measurements and analysed the mass spectrometry data. S.L. performed bioinformatics analyses of the mass spectrometry data. T.F. performed structural modelling. J.R.C., T.B. and A.G. supervised the project and analysed the data. G.S. and A.G. wrote the manuscript with input from all authors.

Competing interests

G.S. and A.G. are inventors on a filed patent application covering the therapeutic targeting of ARD interactions with H4K20me0 for cancer therapy. A.G. and T.F. are co-founders of Ankrin Therapeutics.

Corresponding authors

Correspondence to Till Bartke or Anja Groth.

Integrated supplementary information

  1. Supplementary Figure 1 Domain architecture of TONSL, BARD1 and SLF1.

    Domain boundaries are indicated. TPR, Tetratricopeptide Repeats. UBL, Ubiquitin-Like. LRR, Leucine Rich Repeats. RING, Really Interesting New Gene finger domain. BRCT, BRCA1 C-Terminus.

  2. Supplementary Figure 2 ARD recognition of H4K20me0 recruits BRCA1–BARD1 to post-replicative chromatin.

    a, High-content microscopy of H4K20me0/1/2 levels across the cell cycle. U-2-OS cells were pulsed with EdU, pre-extracted, fixed and processed for immunofluorescence. Cell cycle stages were defined by gating on EdU and DAPI as shown (right). Red bars indicate mean. b, One representative experiment from Fig. 2a is shown. Red bars indicate mean. From left, n = 3357, 2367, 3491, 800, 1721 and 3357, 2367, 3491, 800, 1721. c, Control for equal expression of Flag-HA-BARD1 WT and ARD 3A in inducible U-2-OS cells lines used in Fig. 2b, d, 3a–f. Cells induced or not for Flag-HA-BARD1 WT and ARD 3A expression with TET were fixed directly (without pre-extraction) and stained for HA. Mean HA-BARD1 intensity is indicated relative to HA-BARD1 WT intensity in TET treated G1 cells. Mean with S.D. is shown, n = 3. NS, not significant, ratio paired two-sided t-test. From left, P = 0.3004, 0.3867, 0.9878, 0.2141, 0.9006. d, One representative experiment from Fig. 2b is shown. Red bars indicate mean. From left, n = 3516, 2119, 2495, 953, 1265 and 4642, 2074, 2949, 1198, 1285. e, Control for total expression of BARD1 and BRCA1 in siRNA-treated U-2-OS cells inducible for Flag-HA-BARD1 WT and ARD 3A used in Fig. 2b, d, 3a–f. High-content microscopy of BARD1 and BRCA1 across the cell cycle in siRNA-treated U-2-OS cells induced or not with TET to express Flag-HA-BARD1 WT or ARD 3A. Mean with S.D. is shown, n = 3. Each data point represents the mean of >603 cells. IR treatment: 1 Gy, 2 h recovery before HA staining (right panel). NS, not significant, ratio paired two-sided t-test. f, One representative experiment from Fig. 2d is shown. Mean shown as ‘+’, whiskers indicate 10–90 percentile. From left, (G1) n = 1513, 466, 1203, 478, (G2) n = 685, 551, 626, 495. g, High content microscopy of H4K20me0/1/2 levels in G1 and G2 cells depleted for SET8 as in Fig. 2d. Red bars indicate mean. Cell cycle stages were defined by gating on EdU and DAPI as shown. h, Western blot analysis of H4K20me1/2 levels in SET8 depleted U-2-OS cells. Representative of 2 biological replicates. Unprocessed blots are presented in Supplementary Fig. 5.

  3. Supplementary Figure 3 ARD recognition of H4K20me0 is required for recruitment of BRCA1–BARD1, exclusion of 53BP1 and HR at DSBs in S/G2.

    a, Contribution of RNF8 and H4K20me0 recognition to BRCA1 recruitment at DSBs in late S. High-content microscopy of BRCA1 in siRNA-treated U-2-OS cells induced with TET to express Flag-HA-BARD1 WT or ARD 3A. Cells were fixed 1 h after treatment with IR (5 Gy). The number of BRCA1 foci is shown relative to the control siRNA in Flag-HA-BARD1 WT cells without TET induction. Late S phase cells are defined by gating on EdU and DAPI as in Fig. 2a. Mean with S.D. is shown, n = 3. b, The role of BARD1 ARD recognition of H4K20me0 in antagonizing 53BP1 accumulation. High-content microscopy of IR-induced BRCA1 and 53BP1 foci across the cell cycle in U-2-OS cells induced to express Flag-HA-BARD1 WT or ARD 3A. Left, single cell analysis of BRCA1 and 53BP1 foci and DAPI total intensity. Right, quantification of 4 independent experiments. Foci number is shown relative to BARD1 WT in mid S. Mean with S.D. is shown, n = 4. *P < 0.05, **P < 0.001. NS, not significant, ratio paired two-sided t-test. From left, P = 0.009, 0.0661, 0.0133, 0.0771, 0.0513, 0.0072, 0.0782, 0.0083, 0.0528, 0.0202. c, The HR defect in BARD1 ARD expressing cells is partly rescued by 53BP1 depletion. DR-GFP U-2-OS cells were siRNA treated as indicated and complemented with siRNA-resistant Flag-HA-BARD1 ARD 3A cDNA or empty vector before DR-GFP analysis. HR efficiency is shown relative to control siRNA. Mean with S.D. is shown, n = 3, from left, P = 0.0195 0.0106, ratio paired two-sided t-test. The gating strategy to identify transfected (RFP positive) and HR proficient (GFP positive) cells is shown on the right. d, Complementation of BARD1-null DR-GFP reporter cells with the indicated BARD1 cDNAs as in Fig. 4b, including mouse BARD1 and additional controls. The HR efficiency relative to empty vector (vector, pIRESpuro) is indicated above each bar. The BARD1 domain deletions carry a sequence divergence (Q406)23 from the currently annotated sequence (R406, UniProt ID: Q99728). Importantly, BARD1 Q406 (WT Q406) and BARD1 R406 (WT) were equally proficient in rescuing HR efficiency and for simplicity the sequence divergence is therefore not annotated in Fig. 4b, but indicated here: WT, BARD1 (R406); ARD 3A (R406); WT Q406 (Q406)23; ΔARD (Q406)23; ΔBRCT (Q406)23; mBARD1, mouse BARD1. Mean with S.D., n = 3.

  4. Supplementary Figure 4 Structural modelling of the BARD1 H4K20me0 reader domain and analysis of single point mutations in the ARD.

    a, b, Model of the BARD1 ARD structure (green) in complex with histone H4 (L22-K12) tail (yellow) refined by the Rosetta macromolecular modelling package using the 2.4 Å high-resolution crystal structure of TONSL ARD/H4K20me07 (PDBID 5ja4) as template (see method section for further details). The C-alpha Root-Mean-Square Deviation (RMSD) between the BARD1 ARD model in complex with H3-H4 and the BARD1 ARD crystal structure18 (PDBID 3c5r - in absence of H4) is 0.7 Å. In comparison, the main chain RMSD between the ARD domains of the crystal structure of BARD1 (PDBID 3c5r) and TONSL (PDBID rja4) is 0.9 Å. The hydrogen bonding network of predicted key interactions between the positively charged side chains of H18 and K20 (yellow sticks) and the negatively charged E429, D458, E467 and D500 (green sticks) are illustrated by yellow dashed lines. (b) Structural super positioning of BARD1 ARD/H4 (L22-K12) (green/yellow) with the crystal structure (PDBID 5ja4) of TONSL ARD/H4K20me0 (grey). Repositioning of R17 in H4 is indicated by the dashed black arrow. In the TONSL crystal structure R17 makes interactions with both the main and side chain carbonyl of N571. In the model of the BARD1 ARD in complex with H4, NH2 in the amide side chain of the nearby N504 (which is C608 in TONSL ARD) makes an intramolecular hydrogen bond to the backbone carbonyl of N470 resulting in a relocation of R17. The carbonyl oxygen of the N504 side chain is predicted to make a hydrogen bond interaction with the NH amide backbone atom of K16. c, d, Analysis of BARD1 ARD single amino acid mutants in U-2-OS cells induced to express Flag-HA-BARD1 WT or the indicated mutants. Mean with S.D., n = 3. (c) Total protein levels and chromatin-binding were analysed by high content microscopy in cells either directly fixed or pre-extracted before being processed for HA immunofluorescence. (d) High content microscopy of Flag-HA-BARD1 and BRCA1 IR-induced foci analysed 45 min after IR (3.5 Gy). All constructs carried the Q406 sequence divergence, as described in Supplementary Fig. 3d. e, Alignment of the ARDs of TONSL, BARD1 and SLF1 (from H. sapiens, M. musculus and X. tropicalis). The general ARD consensus sequence is indicated above. The arrows indicate the conserved acidic residues.

  5. Supplementary Figure 5 Unprocessed blots.

    Unprocessed blots for Fig. 1c, d, Fig. 5a, c and for Supplementary Fig. 2h are presented.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–5 and legends for Supplementary Tables 1 and 2.

  2. Reporting Summary

  3. Supplementary Table 1

    Full list of proteins identified by SILAC mass-spectrometry pull-down with H4K20me0 and H4K20me2 di-nucleosomes.

  4. Supplementary Table 2

    Statistics Source Data.

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https://doi.org/10.1038/s41556-019-0282-9