Mechanism of histone H2B monoubiquitination by Bre1

Monoubiquitination of histone H2BK120/123 plays multiple 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 Rad6 E2, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggests a general mechanism of tuning histone specificity via the non-E2-binding RING domain.


Main
Post-translational modification (PTM) of histones plays a central role in regulating eukaryotic transcription. Monoubiquitination of histone H2B-K123 in yeast, K120 in humans (H2B-Ub), is a hallmark of actively transcribed genes that also plays a role in DNA replication, DNA repair and RNA processing 1-3 . H2B-Ub stimulates methylation of histone H3-K4 and K79, and recruits FACT (Facilitates Chromatin Transcription) to promote efficient transcriptional elongation 4,5 . Bre1 is a dimeric ubiquitin E3 ligase that targets the E2 ubiquitin conjugating enzyme, Rad6, to monoubiquitinate H2B-K123 in yeast 6,7 . In humans, the closely related RNF20/RNF40 heterodimer targets RAD6A/B to ubiquitinate histone H2B-K120 8,9 . Mutations and deletions of RNF20/40 are found in a variety of cancers and are indicators of poor prognoses 10 . The mechanism underlying the specificity of H2B-K123/120 ubiquitination is unknown.
We report here the cryo-EM structure of a Bre1 E3 ligase dimer bound to a nucleosome that reveals the molecular basis for specific ubiquitination of histone H2B. The fragment used in the study, Bre1 591-700, includes the RING domain and a coiled-coil that mediates dimerization 11 , and directs specific ubiquitination of H2B-K123 12 . Three distinct states of the Bre1-nucleosome complex were resolved at overall resolutions of 3.47 Å for state 1, 3.25 Å for state 2, and 3.21 Å for state 3 (Extended Data Figs.1,2 and Table 1). In each state, one Bre1 monomer was well ordered, with a local resolution of 3-4 Å, while the other monomer was resolved at resolutions ranging from 4Å to 6Å (Extended Data Fig. 2), indicating somewhat higher mobility. The well-resolved Bre1 density map enabled us to successfully model Bre1 dimer onto nucleosome using its . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint crystal structure 11 . Although the complex contains a polyprotein fusion containing two copies of Bre1 and a C-terminal Rad6 protein joined by flexible linkers, Rad6 is not visible in our cryo-EM maps, presumably due to its weak and transient interactions with the Bre1 RING domain 13 and the nucleosome 14 . Importantly, the fusion enzyme has even higher H2B-ubiquitinating activity on nucleosome as compared to the separated Bre1 and Rad6 enzymes (Extended Data Fig. 3), indicating that the linkers do not interfere with enzyme function.
The Bre1 dimer straddles the periphery of the nucleosome, with one RING domain (Bre1-A) contacting the nucleosome acidic patch and the other (Bre1-B) interacting with the DNA at superhelical position (SHL) 6.5 ( Fig.1a-b). Residues 632-647 form an a helix that mediates coiled coil interactions with the opposing monomer, followed by the catalytic RING domain, which contains a basic patch comprising residues R679, R681, K682, and K688 (Extended Data Fig. 4). The Bre1-A RING domain binds in a similar manner to the nucleosome acidic patch in all three states, with R679 forming salt bridges with H2A residues E61 and D90, and R681 forming salt bridges with H2A residues E61 and E64 (Fig.1b, c). This mode of interaction with the nucleosome acidic patch, termed an "arginine anchor," has been observed in multiple structures 15 . The R679 contact represents a canonical arginine anchor while the R681 contact forms a type 2 variant arginine anchor 15 . The position of the Bre1-B RING relative to the DNA backbone varies more between the three states due to a repositioning of the Bre1 coiled coil. In state 1, the Bre1-B RING is positioned such that residues R679 and R681 are in a position to contact the electronegative DNA backbone (Fig.1d, Extended Data Fig.5a).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made In state 2, the Bre1 dimerization coiled coil is tilted such that the Bre1-B RING rotates by about 10° about the face of the nucleosome and is about 10 Å farther from the DNA.
In state 3, the Bre1 dimer is rotated by an additional 5° on the nucleosome relative to state 2, which shifts the Bre1-B RING by about 5 Å relative to state 2 (Fig.1e, f and Extended Data Fig.5). Importantly, there is a bulge in the DNA in state 3 that repositions the sugar-phosphate backbone 6 Å closer to the Bre1-B RING, placing Bre1-B residue R681 in a position to form electrostatic interactions with the DNA backbone (Fig.1f).
The key roles of Bre1 RING domain residues R679 and R681 in contacting the nucleosome acidic patch and the DNA are consistent with the deleterious effects of substitutions of these residues on Bre1 binding to nucleosomes and H2B ubiquitination activity (summarized in Table S1) . Biochemical studies by Gallego et al. 14 showed that Bre1 substitutions R679D and R681D abrogated nucleosome binding in pull-down assays and decreased H2B ubiquitination activity. Competition assays with the LANA (latency-associated nuclear antigen) peptide, which binds to the nucleosome acidic patch, showed that LANA peptide could compete with Bre1 for binding to the nucleosome acidic patch and reduce H2B monoubiquitination activity, further validates the importance of Bre1 contacts with the nucleosome acidic patch 14 . Importantly, an in vivo study conducted by Turco et al. 12 showed that Bre1 basic patch mutations R675D/R679D and R681D/K682D completely abrogated H2B ubiquitination activity in yeast and resulted in severe growth defects comparable to a bre1 deletion. Bre1 residues R679 and R681 that bind to nucleosome acidic patch are conserved in the . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Fig. 7b-c). Of these, Bre1 substitutions L650A, R675D and K682D abolish H2B monoubiquitination in vitro 14 , while Bre1 mutants L650A and R675D lead to less ubiquitin discharge from Rad6~Ub thioester, consistent with the predicted roles of these Bre1 residues in binding to Rad6 (Table S1) 14 .
A comparison of the Rad6 position in each state indicates that Bre1 alternates between active and inactive, poised states. In state 3, modeling positions the Rad6~Ub thioester directly over histone H2B K120 (corresponding to K123 in the yeast nucleosome), with predicted distances of 2.2 Å between the Sγ of the Rad6 active site cysteine, C88, and Nζ of H2B K120, and 2.8 Å between the carbonyl carbon of Ub G76 and H2B K120 Nζ ( Fig.2a, b). These distances would readily allow ubiquitin transfer from Rad6 to H2B K120. The corresponding distances in state 2 are 5.2 Å and 5.8 Å, respectively . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint (Extended Fig. 8a-b). Interestingly, the modeled position of Rad6 in state 1 places the Rad6~Ub thioester farther away from substrate lysine (8.8 Å and 10.7 Å) (Extended Data Fig. 8c, d). This suggests that each of the three Bre1-nucleosome complex states represents a different Rad6 activity status. Bre1 in state1 and 2 position Rad6 and Ub over the H2B K120 site, presumably in a "poised" state due to the distance from the substrate lysine, while state 3 is likely to facilitate an "active" state that promotes ubiquitin transfer to H2B K120. Interestingly, the histone H2A E3 ligase, BRCA1-BARD1, was also observed to adopt multiple orientations on the face of the nucleosome, with BARD1 forming distinct interactions with the C-terminal α-helix of histone H2B or with the H2B-H4 cleft 18,19 .
Both Bre1 and Ubr1 contain N-terminal helical regions that contact Rad6 and enhance E2 discharge, termed RBD (Rad6 binding domain; residues 1-210) in Bre1 and U2BR (Ubc2-binding region) in Ubr1 12,17 . A recent crystal structure of Bre1-RBD bound to Rad6 revealed that the alpha-helical RBD forms a homodimer that binds to the Rad6 backside 20 . A superposition of the RBD-Rad6 complex with our modelled Rad6 bound to the Bre1-A RING shows that the RBD, which extends away from the nucleosome disk, can be readily accommodated without steric clash with the C-terminal Bre1 dimer or the nucleosome (Extended Data Fig.9). An additional ~440 residues that are not present in the RBD or in the Bre1 fragment in the present study bridge the RBD C-terminus and the Bre1 coiled-coil.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint Mutations in RNF20 and RNF40, have been found in a variety of cancers according to the cancer genomics database of cBioPortal (http://www.cbioportal.org). Of these, 27 map to residues in the RING domain or the a helical regions of RNF20 and RNF40 (Tables S2 and S3; Extended data Fig. 6a-b). RNF20 residue R955, corresponding to Bre1-R681 that binds the nucleosome acidic patch, is mutated to histidine in esophagogastric cancer (Extended data Fig.6a-b and Table S2). In addition, substitutions of RNF20 residues C924, R949 and Q954, which correspond to Bre1 residues L650, R675 and M680 that are predicted to interact with Rad6, are implicated in multiple cancers. Substitutions in these RNF20 residues that are predicted to disrupt the canonical E3 RING-E2 interface occur in non-small cell lung cancer (C924F, R949L, Q954*), cervical cancer (R949C) and colorectal cancer (R949H) (Extended data Fig.6ab and Table S2).
A comparison of the Bre1 complex with that of E3 ligases that ubiquitinate other histone residues points to a pivot-like mechanism for tuning E3 ligase specificity. The positioning on the nucleosome of Bre1, which ubiquitinates H2B-K120/123, is markedly different from that of the heterodimeric E3 ligase, Ring1B/Bmi1 21 , which ubiquitinates H2A-K119, and BRCA1/BARD1 19 , which ubiquitinates histone H2A K125/127/129. Like Bre1, the RING domain that recruits the E2, Ring1B or BRCA1, binds to the nucleosome acidic patch with basic residues (Fig.2c-e). It is the orientation of the second RING domain in each complex that governs the positioning of the E2, and hence its specificity. Whereas Bre1 uses the second RING domain to interact with the DNA, Bmi1 and BARD1, respectively, bind to different locations in the globular histone . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint core. Bmi1 caps the C-terminal end of H3 α1 helix via salt bridges formed by K62 and R64 and acidic residues in histones H3 and H4 21 , while BARD1 flexibly binds to the nucleosome H2B αC-helix 18 or H2B/H4 cleft with its Trp91 inserted 19 . These second RING domains thus play the defining role in determining the E3 ligase specificity for its histone substrate.

Acknowledgements
We thank Johns Hopkins colleagues Chuan Liu and Edward Twomey for advice on

Competing Interests
The authors declare no competing interests.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made      . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint Extended Data Fig.6 RNF20/40 mutation sites mapped to RING domain and preceded ahelix 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.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint 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.
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After plasmid extraction, the 601 DNA was excised with EcoRV, and recovered as described previously 23 . Nucleosomes were reconstituted from purified histones and DNA as previously described 23 .

Cryo-EM sample preparation
A sample of 100 nM unmodified nucleosome was incubated with 10 µM Bre1-Bre1-Rad6 fusion protein in cross-linking buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM DTT) at room temperature for 30 min. An equal volume of 0.15% glutaraldehyde was then added to the sample mixture. After incubation on ice for 1 hour, the cross-linking reaction was quenched by the addition of 100 mM Tris pH 7.5. The sample was dialyzed overnight into quenching buffer (50 mM Tris pH 8.0, 50 mM NaCl, 1 mM DTT) and applied to a Superdex 200 10/300 size exclusion column (GE Healthcare) that was pre-equilibrated with cross-linking buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM DTT). The peak fractions were concentrated to a final sample concentration of 1 mg/mL. Quantifoil R2/2 copper 200 mesh grids (Electron Microscopy Sciences) were glowdischarged for 45 seconds at 15 mA, after which, 3 µl of cross-linked sample was . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint applied to the grids and blotted for 3.5 seconds with a blot force of 5, then immediately plunged frozen in liquid ethane using a Vitrobot Mark IV apparatus (Thermo Fisher Scientific) at 4°C, 100% humidity. The dataset was processed using cryoSPARC 24 . After patch-based motion correction and contrast transfer function estimation, 3,778 micrographs were selected for particle picking. Initial particles were picked by Blob picker. After particle inspection and extraction, a total of 721,361 initial particles were used in 2D classification. The best 2D classes were then selected as templates for template picking of particles. Then, a total of 2,134,610 particles were extracted and subjected to 2D classification, yielding a particle set of 1,418,167 particles. After initial reconstruction and heterogeneous refinement with 4 classes, one 3D class accounting for 534,058 particles was generated and refined. The refined particles stack was re-extracted for further heterogeneous refinement with 4 classes, among which two classes (435,496 particles) were combined for non-uniform refinement. Based on this EM map, a mask encompassing one Bre1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Cryo-EM data processing and model building
The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint dimer and less than half of nucleosome was generated. After C2 symmetry expansion of particles, a total of 870,992 particles were subjected to 3D classification with the above mask. Among the ten 3D classes, five classes had well-resolved Bre1 dimer, and three states of Bre1 binding to nucleosome were identified. The three state maps were further refined to a resolution of 3.47 Å for state 1, 3.25 Å for state 2, and 3.21 Å for state 3. Finally, a sharpening B factor of -5 Å 2 was applied to maps of state1, 2 and 3.
We note that, although Rad6 was fused to the Bre1 C-terminus in an effort to overcome the low affinity of Rad6 for the Bre1 RING domain 12,13 , there was no density corresponding to Rad6 in any of the maps. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 28, 2023. ; https://doi.org/10.1101/2023.03.27.534461 doi: bioRxiv preprint immunoblotting with Ubiquityl-histone H2B K120 antibody (1:2500 dilution, CST #5546).
The atomic models are available in the Protein Databank (PDB) database with PDB ID 8T3Y (state 1), 8T3W (state 2) and 8T3T (state 3).
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