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BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1

Abstract

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

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Fig. 1: RING E3 ligases ubiquitylate H2A at distinct sites on the nucleosome.
Fig. 2: Cryo-EM structure of the BRCA1-UbcH5c/BARD1/nucleosome complex.
Fig. 3: BRCA1/BARD1 RING-histone interactions.
Fig. 4: Comparison to the Ring1b-UbcH5c/Bmi1/nucleosome complex.
Fig. 5: Lysine position along the flexible C-terminal tail of H2A dictates ubiquitylation efficiency.
Fig. 6: H2A C-terminal tail dynamics in E3-E2/nucleosome complexes.
Fig. 7: Enhanced nucleosome ubiquitylation activity and binding of full-length BRCA1/BARD1.

Data availability

The cryo-EM map of the BRCA1-UbcH5c/BARD1/nucleosome complex has been deposited to the Electron Microscopy Data Bank under accession code EMD 22581 and the atomic model to the Protein Data Bank under accession code 7JZV. NMR chemical shift assignments were deposited to the Biological Magnetic Resonance Data bank under accession code 50604. Plasmid reagents generated in this study can be obtained upon request from the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank S. Tan (Penn State University) for sharing Ring1b/Bmi1plasmids and L. Kay (University of Toronto) for sharing the 153-base-pair Widom 601 repeat plasmid. We thank N. Zheng for his insightful feedback on the manuscript and sharing laboratory equipment; T. Hinds for assistance with ITC experiments; P. Hsu for advice on nucleosome complexes; J. Quispe and Q. Beedle for technical assistance with cryo-EM data collection; and L. Walls, A. Borst and D. Veesler for advice on single-particle cryo-EM data processing. S.R.W. acknowledges support from the NIH (T32GM008268). A.L.B. was supported by the NIH (T32GM008268 and 1F31EY030732). D.P.F. was supported through the Human Frontiers Science Program (RGP0061/2019). J.M.H. was supported by the NIH (T32GM007270). W.Z. was supported by a V Scholar Grant (V2019.Q13) from the V Foundation and a Young Investigator Award from the Max and Minnie Tomerlin Voelcker Fund. F.D. was supported by the NIH (GM123089). R.E.K. is the Edmond H. Fischer/Washington Research Foundation Endowed Chair in Biochemistry and is supported by the NIH (GM088055).

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Contributions

S.R.W., M.D.S., P.S.B. and R.E.K. conceived the project, designed the experiments and analyzed data. S.R.W. prepared the cryo-EM sample, imaged and analyzed data with A.L.B. and J.M.H. under the supervision of J.M.K. D.P.F. built the atomic model under the supervision of F.D. with input from S.R.W. J.K. generated methylated nucleosomes under the supervision of C.C. S.R.W. and L.M.T. conducted and analyzed the NMR experiments. M.W. and W.Z. produced full-length BRCA1/BARD1 and performed binding assays. S.R.W. and A.P. purified proteins, performed assays and analyzed data. S.R.W., P.S.B. and R.E.K. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Rachel E. Klevit.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Specificity of BRCA1/BARD1-dependent nucleosome ubiquitylation and validation of the E3-E2 chimera.

a, Western blot analysis of all four histone subunits from the same nucleosome ubiquitylation reaction. b, Native-gel EMSA measuring NCP binding of BRCA1-UbcH5c/BARD1 constructs with various Gly/Ser-repeat linker lengths. The E3-E2 chimera with a seven-residue linker was used for structure determination and all subsequent experiments. c, Nucleosome ubiquitylation assays using BRCA1/BARD1 with indicated E2 enzymes. d, Coomassie-stained gel under reducing or non-reducing conditions of an E2 charging reaction using the BRCA1-UbcH5c/BARD1 chimera. e, Nucleosome ubiquitylation activity of BRCA1/BARD1 with UbcH5c in trans or the BRCA1-UbcH5c/BARD1 chimera with wild-type (WT) or Lys125/127/129Arg (3KR) NCP substrates. Data in panels c and e are representative of n=2 independent experiments. Uncropped gels/blots in panels a-e are available as source data. Source data

Extended Data Fig. 2 Purification of a stable BRCA1-UbcH5c/BARD1/nucleosome complex.

a, Coomassie-stained gel of an E2 charging reaction using UbcH5cC85K and BRCA1-UbcH5cC85K/BARD1. b, Size exclusion chromatography (SEC) of NCPs (black) with excess BRCA1-UbcH5c/BARD1 (purple) or BRCA1-UbcH5cC85K/BARD1 (green) with 35 mM NaCl in SEC buffer. c, Coomassie-stained gels of fractions from SEC binding experiments shown in panel b. d, SEC of NCPs with excess BRCA1-UbcH5cC85K/BARD1 with 100 mM NaCl (black) or 25 mM NaCl (green) in SEC running buffer. e, Coomassie-stained gels of fractions from SEC binding experiments shown in panel d. f, Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of NCPs (black) and the BRCA1-UbcH5cC85K/BARD1/NCP complex (green). Dashed lines report MALS molecular weight (MW) data. The MW value reported is the average MW from light scattering ± 1-s.d. where error is a measure of statistical consistency of light scattering data and not an absolute bound on the error of MW. The expected MW is 203.7 kDa for the NCP and 287.2 kDa for the complex bound with 2:1 stoichiometry. Uncropped gels in panels a, c, and e are available as source data. Source data

Extended Data Fig. 3 Cryo-EM processing workflow.

a, Flow chart of data processing steps. Objects with dark borders represent solvent masks and are not density maps. b, Representative motion-corrected micrograph from data set. White scale bar is 100 nm.

Extended Data Fig. 4 Cryo-EM validation and example density.

a, Local resolution estimate from CryoSparc after non-uniform refinement at the FSC=0.143 cutoff plotted on the final density modified map of the whole complex (left) and close-up on the RING-histone interface (right). b, Half-map Fourier shell correlation curves from CryoSparc non-uniform refinement (red) and after density modification (blue). c, Euler angle distribution of refined particle subset used in final cryo-EM reconstruction. d, Main chain trace of the BRCA1 RING, BARD1 RING, four-helix bundle, and UbcH5c fit into the cryo-EM map. The active site position of UbcH5c (Lys85) Cα is shown as a sphere. e, Subset of important histone regions fit into the cryo-EM map with side chains shown (except for H2A C-terminal tail).

Extended Data Fig. 5 Extensive analysis of the BRCA1/BARD1 RING-histone interface.

a, b, c, Close-up view of BRCA1 and BARD1 RING domains in the complex showing side chains of residues mutated in nucleosome ubiquitylation assays in panels d-g and j-k, d, Representative nucleosome ubiquitylation assay using the indicated BRCA1 RING mutants. e, Quantified nucleosome ubiquitylation assays for BRCA1 mutants shown in panel d. (f, g) Same experimental set-up as panels d and e using the indicated BARD1 mutants. h, ITC binding data for BRCA1/BARD1 and indicated mutants with NCPs. Summarized data are also shown in Fig. 3e. i, 1H15N-TROSY-HSQC spectra of wild-type (WT) BRCA1/BARD1 and BRCA1(WT)/BARD1(Trp91Ala). j, k, Same experimental set-up as panels d and e using BARD1 mutants reported on the COSMIC database. l, E2~Ub lysine discharge assay using the indicated BRCA1/BARD1 mutants. m, Quantified E2~Ub lysine discharge assays using the BRCA1/BARD1 mutants shown in panel l. n, o, Same experimental set-up as panels l and m using BARD1 COSMIC mutants. Quantified nucleosome ubiquitylation assay data show the mean and error bars are ± 1-s.d. of n=3 independent experiments at 10-minute (gray bars) and 30-minute (colored bars) time points. Statistical analysis is compared to wild-type at the 10-minute time point. Quantified E2 lysine discharge data show the mean and error bars are ± 1-s.d. of n=3 independent experiments at 2-, 6- and 10- minute time points (light, medium, and dark gray bars). Statistical comparisons are indicated with lines above the graphs. All p-values were calculated using a two-tailed Student’s t-test (* p≤0.05, ** p≤0.005, ns = not significant). Uncropped gels/blots in panels d, f, j, l, and n and data from graphs in panels e, g, k, m, and o are available as source data. Source data

Extended Data Fig. 6 Comparison of nucleosome requirements for H2A-modifying E3s.

a, Comparison of the conserved BRCA1 and Ring1b arginine anchor motif interactions (PDB: 4R8P). Complexes were aligned by H2B on the bound face of the NCP. H2A from the Ring1b-UbcH5c/Bmi1 nucleosome complex is shown in gray. b, Nucleosome ubiquitylation assays using the indicated H2A/H2B acidic patch NCP mutants with BRCA1/BARD1 (top) and Ring1b/Bmi1 (bottom). c, Comparison of interactions between the RING domains of BRCA1/BARD1 and Ring1b/Bmi1 with the H2B αC helix residues assayed in panels d and e. d, Representative nucleosome ubiquitylation assays using the indicated H2B αC helix NCP mutants with BRCA1/BARD1 (top) or Ring1b/Bmi1 (bottom). e, Quantified assays using the NCP mutants and E3s from panel d. f, g, Same experimental set-up as in panels d and e using H2B/H4 cleft NCP mutants. h, BARD1-histone binding interface with side chain density for H3 Phe78 and Lys79 shown as semi-transparent surface. i, j, Same experimental set-up as panels d and e using H3 Lys79Ala NCP mutant. k, l, m, Representative nucleosome ubiquitylation assays using H3 Lys79me1 and Lys79me2 mimetic NCP substrates with the indicated E3s. n, o, p, Quantified assays using H3 Lys79 methylation mimetic nucleosome substrates and the indicated E3s. Quantified ubiquitylation assays show the mean and error bars are ± 1-s.d. of n=3 independent experiments (except panel g where n=6 for wild-type) plotted for 10-minute (gray bars) and 30-minute (colored bars) time points. P-values for each mutant were calculated using a two-tailed Student’s t-test compared to wild-type (* p≤0.05, ** p≤0.005, ns = not significant). Panels g (top), and n are also shown in Fig. 3. Uncropped blots in panels b, d, f, i, k, l, and m and data from graphs in panels e, g, j, n, o, and p are available as source data. Source data

Extended Data Fig. 7 Comparison to the unbound RING heterodimer and Ring1b-UbcH5c/Bmi1/nucleosome complexes.

a, Structural alignment of the BRCA1/BARD1 RING heterodimers from the NCP complex and the unbound structure (PDB: 1JM7, NMR ensemble model 1) aligned by the four-helix bundle. b, Structural alignment of the NCP from the indicated complexes by H2B on the E3-E2-bound side. c, Structural alignment of BRCA1-UbcH5c and Ring1b-UbcH5c from E3-E2/NCP complexes. d, Comparison of BRCA1-bound and Ring1b-bound UbcH5c in NCP complexes showing side chains mutated in panels g and h. Some atoms for Arg72 and Lys128 are not modelled in the Ring1b-bound structure. Green spheres are Cα of H2A Lys118. e, Analysis of NCP binding by the BRCA1/BARD1 RING heterodimer and the BRCA1-UbcH5c/BARD1 (WT UbcH5c active site) chimera by ITC. Data are representative of n=2 experiments. f, Comparison of NCP binding strength for BRCA1/BARD1 and Ring1b/Bmi1 RING heterodimer and E3-E2 chimeras. Affinities for BRCA1/BARD1 were determined using ITC while affinities for Ring1b/Bmi1 were measured using a fluorescence based binding assay27. g, Representative nucleosome ubiquitylation assays using BRCA1/BARD1 or Ring1b/Bmi1 with indicated UbcH5c mutants. h, Quantification of nucleosome ubiquitylation using the UbcH5c mutants and E3s from panel g. Data show the mean and error bars are ± 1-s.d. of n=3 independent experiments plotted for 10-minute (gray bars) and 30-minute (colored bars) time points of the reactions. P-values for each mutant were calculated using a two-tailed Student’s t-test compared to wild-type (* p≤0.05, ** p≤0.005, ns = not significant). Uncropped blots in panel g and data from graphs in panel h are available as source data. Source data

Extended Data Fig. 8 NMR analysis of E3-E2/nucleosome complexes.

a, 1H15N-TROSY-HSQC spectrum of 2H15N13C-H2A in NCP (~180 µM sample) with residue assignments plotted. Signals with asterisks were not able to be assigned. Residues 120# and 120## appear to be alternate confirmations of 120 based on Cα chemical shifts of residues 119 and 120 in the HNCA and HNCOCA spectra. Assignment of the minor population for 6# was based on the similar back bone H, N, and Cα chemical shifts as compared to the major residue 6 population. b, Sequence of H2A N- and C-terminal tails observable in NMR spectrum in panel a with assigned residues bolded. c, Overlay of 1H15N-TROSY-HSQC spectra of 2H15N-H2A in NCP, with BRCA1-UbcH5cC85K/BARD1 added at increasing concentrations. d, Quantification of signal broadening from spectra in panel c, as a function of BRCA1-UbcH5cC85K/BARD1 added, normalized to the first titration point (60 μM addition). Signal behavior clusters into two groups, where resonances from the extreme N- and C- terminal tails lose signal at a similar rate (cluster 1). e, Overlay of 1H15N-TROSY-HSQC spectra of 2H15N-H2A in NCP (black, bottom), with BRCA1-UbcH5cC85K/BARD1 added (red, middle), or BRCA1-UbcH5c/BARD1 (blue, top). f, Quantification of intensity for H2A signals from spectra in panel e comparing the bound complexes to the apo reference spectrum (Icom/Iref). g, Overlay of 1H15N-TROSY-HSQC spectra of 2H15N-H3 in NCP (black, bottom), with BRCA1-UbcH5cC85K/BARD1 added (red, middle), or Ring1b-UbcH5c/Bmi1 (pink, top). h, Quantification of intensity for H2A signals from spectra in panel g comparing the bound complexes to the apo reference spectrum (Icom/Iref). Concentrations of E3-E2 complexes added as well as labelled NCPs in the experiment are indicated in the figure. Data from graphs in panels d, f, and h are available as source data. Source data

Extended Data Fig. 9 Biochemical purity and H2A specificity of full-length BRCA1/BARD1.

a, Coomassie-stained gel of purified full-length BRCA1/BARD1. b, Nucleosome ubiquitylation assay using full-length BRCA1/BARD1 and wild-type (WT) or H2A Lys125/127/129Arg (3KR) nucleosome substrates. Uncropped gels/blots in panels a and b are available as source data. Source data

Extended Data Fig. 10 Compatibility of closed E2~Ub conformations.

a, Structural overlay of the BRCA1-UbcH5c/BARD1/nucleosome complex with indicated RING/E2-Ub crystal structures aligned to the RING domain of BRCA1. b, Comparison of UbcH5c location in the nucleosome complex (purple) and indicated reported RING/E2-Ub structures from panel a with cryo-EM density for UbcH5c shown as gray volume. Density for the E2 is shown from a map that was locally filtered in CryoSparc to decrease excessive noise introduced from map sharpening in this region.

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Witus, S.R., Burrell, A.L., Farrell, D.P. et al. BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1. Nat Struct Mol Biol (2021). https://doi.org/10.1038/s41594-020-00556-4

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