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Mechanism of histone H2B monoubiquitination by Bre1

Nature Structural & Molecular Biology volume 30, pages 1623–1627 (2023)Cite this article

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Abstract

Monoubiquitination of histone H2B-K120/123 plays several roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase Bre1 reveals that one RING domain binds to the nucleosome acidic patch, where it can position the E2 ubiquitin conjugating enzyme Rad6, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggest a general mechanism of tuning histone specificity via the non-E2-binding RING domain.

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Fig. 1: Cryo-EM structures of Bre1 dimer bound to nucleosome reveal several conformations.
Fig. 2: Bre1 orients Rad6 to ubiquitinate histone H2B-K120.

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Data availability

The maps are available in the Electron Microscopy Data Bank (EMDB) database under accession no. EMD-41016 (state 1), EMD-41015 (state 2) and EMD-41011 (state 3). The atomic models are available in the Protein Data Bank database: PDB 8T3Y (state 1), PDB 8T3W(state 2) and PDB 8T3T (state 3). Source data are provided with this paper.

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Acknowledgements

We thank Johns Hopkins colleagues C. Liu and E. Twomey for advice on Cryo-EM data collection and processing and X. Zhang for valuable suggestions on constructing plasmids. Supported in part by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract 75N91019D00024. This work was supported by National Institute of General Medical Sciences grant GM130393 (C.W.).

Author information

Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Fan Zhao, Chad W. Hicks & Cynthia Wolberger

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  1. Fan Zhao
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  2. Chad W. Hicks
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  3. Cynthia Wolberger
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Contributions

C.W. and F.Z. conceived and designed the study. F.Z. and C.W.H. performed experiments and processed data. F.Z. carried out modeling and map interpretation with assistance from C.W. F.Z., C.W.H. and C.W. wrote the manuscript. C.W. supervised the study.

Corresponding author

Correspondence to Cynthia Wolberger.

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The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Uhn-Soo Cho and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Sara Osman and Carolina Perdigoto were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cryo-EM processing workflow.

The red dashed line indicates the mask generated for use in 3D classification. Three states of Bre1–nucleosome complex are identified and refined respectively.

Extended Data Fig. 2 Cryo-EM validation and example density.

a–c, Local resolution estimation and gold-standard Fourier shell correlation (GSFSC) curves at a FSC cutoff of 0.143 for each state of Bre1–nucleosome complex. d, Representative cryo-EM density maps for the histones, DNA, and Bre1.

Extended Data Fig. 3 Comparison of enzymatic activity of fused and separated Bre1 and Rad6 enzyme.

In vitro nucleosome H2B ubiquitination assay was performed with fused or separated Bre1 and Rad6 enzyme. Samples were quenched by SDS-loading buffer at indicated timepoints and subjected to immunoblotting. Data is representative of 2 independent experiments.

Source data

Extended Data Fig. 4 Electrostatic surface representation view of Bre1 dimer (PDB:4R7E).

Bre1 is shown in a cartoon representation with basic residues highlighted as sticks.

Extended Data Fig. 5 Multiple conformations of the Bre1–nucleosome complex.

a–c, Cryo-EM maps of the Bre1–nucleosome complex with an atomic model fitted to states 1, 2 and 3. d, Superimpose of Bre1–nucleosome complex maps comparing states 1, 2 and 3. White arrows show the changes between each conformational state.

Extended Data Fig. 6 RNF20/40 mutation sites mapped to RING domain and preceded α-helix in cancer genomics.

a, Sequence alignment of coiled coil and RING domains of Bre1, RNF20 and RNF40. Black triangle indicates the mutation sites of RNF20 and RNF40 in cancer genomics. b, Complex structure model of Bre1 RING-Rad6-nucleosome. Bre1 residues corresponding to RNF20/40 mutated sites were shown as sticks with dot sphere.

Extended Data Fig. 7 Canonical interface between E3 RING and E2.

a, Superposition of multiple E2-E3 structures. E2-E3 complex structures of RNF4-UbcH5a (PDB: 4AP4), Ubr1-Rad6 (PDB: 7MEX), Ring1b-UbcH5c (PDB: 4R8P), BRCA1-UbcH5c (PDB: 7LYB) and TRIM25-UbcH5 (PDB: 5FER) are aligned together. b, The modeled structure of Bre1 RING-Rad6. c, Residues located in modeled Bre1A RING-Rad6 interface. The side chains of residues located in the interface are shown as sticks.

Extended Data Fig. 8 Superposition of Bre1–nucleosome complex (state 2 and 1) and Ubr1 RING-Rad6-ubiquitin (PDB: 7MEX).

a, c, Ubr1 RING domain is aligned to Bre1-A RING domain in state 2 (a) and state 1 (c). b, d, Close-up view of Rad6 active site C88, ubiquitin G76, and H2B K120 in state 2 (b) and state 1 (d). The H2B K120 rotamer with the shortest distance from Rad6 C88 and ubiquitin G76 is shown here.

Extended Data Fig. 9 Structural alignment of Bre1 RBD-Rad6 complex and Bre1 RING-Rad6-Ub-nucleosome complex (model of state 3).

a, b, Rad6 in Bre1 RBD-Rad6 complex is aligned to the Rad6 in Bre1 RING-Rad6-Ub-nucleosome complex (model of state 3).

Supplementary information

Source data

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Zhao, F., Hicks, C.W. & Wolberger, C. Mechanism of histone H2B monoubiquitination by Bre1. Nat Struct Mol Biol 30, 1623–1627 (2023). https://doi.org/10.1038/s41594-023-01137-x

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