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. 2024 Mar 22;15(1):2580.
doi: 10.1038/s41467-024-46910-8.

Structure of the human Bre1 complex bound to the nucleosome

Shuhei Onishi  1 , Kotone Uchiyama  1 , Ko Sato  1 , Chikako Okada  1 , Shunsuke Kobayashi  1 , Keisuke Hamada  1 , Tomohiro Nishizawa  2   3 , Osamu Nureki  2 , Kazuhiro Ogata  4 , Toru Sengoku  5
Affiliations

Structure of the human Bre1 complex bound to the nucleosome

Shuhei Onishi et al. Nat Commun. .

Abstract

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0-6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overall structure.
a Domain structures of yeast Bre1 and human Bre1A and Bre1B. An N-terminal helical domain (HD) and two coiled-coil regions (CC1 and CC2) are predicted based on the model structures calculated using AlphaFold. The residue numbers of the domain boundaries are shown. The arrows indicate the regions predicted to form intermolecular interactions. b Cryo-EM density map in two views, colored in accordance with the model in (c). c, d Atomic models of the two RING domains of Bre1A and Bre1B bound to the nucleosome in two views. In (c) (Model I) and (d) (Model II), RINGA and RINGB bind to the acidic patch, respectively. e Structure of the RING domain heterodimer (two views). Six basic residues that are important for nucleosome binding are shown. Zinc ions are shown as a sphere model. f Electrostatic surface potential of the RING domain heterodimer (two views). g Electrostatic surface potential of the nucleosome. The two Bre1-interaction interfaces (the acidic patch and the DNA backbone near SHL6.0) are indicated by green circles.
Fig. 2
Fig. 2. Flexible binding of the Bre1 complex revealed by the 3DFlex analysis.
a Distribution of the latent coordinates. Coordinates that correspond to the structures shown in (b–e) are indicated. Green and orange circles represent the volume series shown in Supplementary Movies 2 and 3, respectively. b–e Conformational changes represented by the first (b, c) and second (d, e) dimensions. b, d An overall view. c, e A close-up view. f Residues whose modifications or substitutions (H2BS112GlcNAc, H3K36M, H3K37ac, H3Y41ph, H3R42A, H3K56ac, and H3R42me2a) upregulate Bre1 activity. H2BK120 itself is also shown.
Fig. 3
Fig. 3. Interactions between the Bre1 complex and the nucleosome.
a Structure of Model I. The magnified regions in (b, c) are indicated. b Interactions between RINGA and the acidic patch in Model I. Hydrophilic interactions (salt bridges and hydrogen bonds) are indicated by gray dotted lines. c Basic residues of RINGB near the DNA phosphates in Model I. d A schematic representation of the protein constructs used in the experiment shown in (e). TS, Twin-Strep-tag. e H2BK120 ubiquitination assay of the wild type (WT) and mutants possessing substitutions at basic residues. The signals were normalized usig the normalization control. The mean and standard deviation of six (for WT) or three (for mutants) independent results are shown. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Identification of Bre1A residues likely to mediate Rad6A binding.
a Structural modeling of the RINGA-RINGB-Rad6A complex. The current structure of the RINGA-RINGB complex is superposed on that of the RNF4 dimer bound to UbcH5A and ubiquitin (PDB ID 4AP4, ubiquitin not shown), and then the structure of Rad6A (PDB ID 6CYO) is further superposed on that of UbcH5A. b RINGA residues that are not conserved in RINGB. The RINGA structure is shown as a surface representation, and 10 non-conserved residues are colored red. Three residues near a possible Rad6A binding region are labeled. c Close-up view of the RINGA-RINGB-Rad6A model in (b). d H2BK120 ubiquitination assay of the wild type (WT) and mutants possessing substitutions at the possible Rad6A binding region. The constructs shown in Fig. 2d and their mutants were used. GA: T948GA-T952AA. TT: G974TB-A978TB. The mean and standard deviation of six (for WT) or three (for mutants) independent results are shown. Source data are provided as a Source Data file. e The binding affinities of the WT and mutants for the nucleosome. The constants between the Bre1 complexes and the nucleosome are shown with their 1-sigma confidence interval. The values were calculated with four (for GA + TT) or three (for the others) independent results.
Fig. 5
Fig. 5. A model for H2BK120-specific ubiquitination by the Bre1 complex.
a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and H2B. Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RINGA, but not RINGB, can recruit Rad6A and ubiquitin. Bre1B with G974TB-A978TB double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.
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