Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome

Abstract

The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the human Ring1B–Bmi1–UbcH5c E3–E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with several nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.

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Figure 1: Crystal structure of PRC1 ubiquitylation module–NCP complex.
Figure 2: Ring1B–Bmi1 E3 heterodimer interacts with histone surface.
Figure 3: UbcH5c binds to nucleosomal DNA enhancing Ring1B–Bmi1–NCP affinity.
Figure 4: Model of ubiquitylation transition state complex.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structure have been deposited with the Protein Data Bank under accession code 4R8P.

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Acknowledgements

We would like to thank the staff of APS NE-CAT beamlines 24ID-E and 24ID-C for their assistance during synchrotron data collection; H. Yennawar, N. Yennawar and J. Fecko at the Penn State Huck Institute X-ray core facility; A. Minns, M. Moore and J. Malloy for reagent preparation; T. Girish and J. Huang for assistance with data collection; S. Wang for providing U2OS cDNA; R. Makde and members of the Tan laboratory and the Penn State Center for Eukaryotic Gene Regulation for discussions. This work was supported by NIGMS grants GM060489-09S1, GM088236 and GM111651 to S.T. R.K.M. is supported by a Damon Runyon Post-doctoral fellowship (DRG 2107-12).

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Contributions

R.K.M. designed the study, cloned, purified and crystallized macromolecules, collected and processed X-ray data, refined and analysed the structure, performed nucleosome ubiquitylation and binding assays, and wrote the paper. R.C.H. cloned, purified and crystallized macromolecules and performed nucleosome ubiquitylation assays. S.T. designed the study, cloned and purified macromolecules, collected X-ray data, analysed the structure and edited the paper. All authors commented on the manuscript.

Corresponding author

Correspondence to Song Tan.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Alignment of PRC1 ubiquitylation module and NCP with previously determined sub-structures and comparison of the two halves of the structure.

a, Alignment of PRC1 ubiquitylation model from the proximal (darker colours) and distal (lighter colours) halves of the PRC1 ubiquitylation module–NCP structure with the previously determined structure of the PRC1 ubiquitylation module alone22 (grey, PDB code 3RPG) using all backbone atoms. b, c, A similar alignment using the Ring1B–Bmi1 subcomplex (b) or UbcH5c only (c). d, Alignment of the NCP from the PRC1 ubiquitylation module–NCP core particle complex (coloured) and the previously determined NCP complex25 (PDB code 3LZ0) containing the identical 601 nucleosome positioning sequence. e, R.m.s.d. for each of the alignments calculated over all backbone atoms. f, g, Orthogonal views of an alignment of two copies of the complex following rotation of one copy about the pseudo-two-fold axis of symmetry of the nucleosome. This was accomplished by simultaneously aligning each histone in one copy to its symmetry related counterpart in the other copy. For visualization, the PRC1 ubiquitylation module from one copy of the structure is depicted with lighter colours. h, Zoomed view of the alignment showing PRC1 ubiquitylation module–NCP interface. The proximal and distal PRC1 ubiquitylation modules are shown in dark and light colours, respectively. Hinge indicated by arrows. i, R.m.s.d. for each of the components following alignment of the two halves as described above. j, Buried surface area calculations for the indicated interfaces on the proximal and distal sides of the structure.

Extended Data Figure 2 Characterization of fused and unfused UbcH5c.

a, Coomassie-stained gel of E1-mediated ubiquitin transfer to UbcH5c(Cys85Lys) mutant (UbcH5c) alone and in the context of the fused PRC1 ubiquitylation module. Ubiquitin is transferred by the E1 Uba1 to the UbcH5c mutant in an ATP-dependent manner, black arrow (lanes 1 and 2). No ubiquitin transfer to the fused PRC1 ubiquitylation module is observed (lanes 3 and 4, expected band position for Ring1B–UbcH5c–ubiquitin conjugate indicated by a blue arrow). The Cys85Lys mutant was used to assist in visualization of the UbcH5c–ubiquitin stable isopeptide conjugates. b, Ring1B of the fused PRC1 ubiquitylation module (blue) clashes with ubiquitin in the E1 Uba1 adenylation site (green). Alignment of UbH5c subunit of PRC1 ubiquitylation module22 (PDB code 3RPG) to Ubc4 in Ubc4–Uba1–ubiquitin structure24 (PDB code 4II2). Ubc4 and Uba1 are shown in pink and grey, respectively. c, Intrisic activity of wild-type and mutant UbcH5c enzymes. Time-course of UbcH5c-ubiquitin thioester aminolysis after lysine addition. Reactions quenched with non-reducing loading buffer unless 100 mM DTT addition is indicated (dagger). Asterisk denotes a non-reducible product.

Extended Data Figure 3 Comparison of the proximal and distal Ring1B–, Bmi1– and UbcH5c–nucleosome interfaces.

ac, Identical views of Ring1B–Bmi1 saddle from proximal (a) and distal (b) halves of structure and overlay (c). dg, Ring1B–histone interface views, including zoom out (d) with box indicating field of view depicted in magnified panels from proximal (e) and distal (f) halves of structure and overlay (g). hk, Similar views of Bmi1–histone interface. ls, Similar views of UbcH5c–DNA interfaces. tu, 2mFo − DFc difference Fourier transform electron density maps for the Ring1B–nucleosome interface contoured at 1.0σ on the proximal (t) and distal (u) sides of the PRC1 ubiquitylation module–NCP structure. v, w, Similarly contoured electron density map for the proximal (v) and distal (w) Bmi1–nucleosome interface. All views are identical to those depicted in Figs 2 and 3.

Extended Data Figure 4 Chromatin factors use an arginine-anchor to bind the H2A–H2B acidic patch.

ae, Identical views of E3 ligase Ring1B (blue) (a), viral LANA peptide (purple, PDB code 1ZLA; ref. 27) (b), chromatin factor RCC1 (purple, PDB code 3MVD; ref. 28) (c), centromeric protein CENP-C (green, PDB code 4INM; ref. 30) (d) and silencing protein Sir3 (brown, PDB code 3TU4; ref. 29) (e) each bound to the acidic patch. Conserved arginine is shown in space-filling representation. Other arginines involved in binding are shown as sticks. UbcH5c and the Ring1B loop between residues 81 and 98 are not shown for figure clarity.

Extended Data Figure 5 Ring1B–Bmi1- and BRCA1–BARD1-mediated ubiquitylation of nucleosomes containing histone mutations.

a, b, Coomassie-stained gels of ubiquitylation assays using nucleosomes with the specified histone mutants, E1 Uba1, E2 UbcH5c, STR-His10 tagged ubiquitin and Ring1B–Bmi1 (a) or BRCA1–BARD1 RING heterodimers (b). Tagged H2B (STR-His6) was used to prevent electrophoretic comigration with unmodified H2A. Experiment was repeated twice.

Extended Data Figure 6 Fluorescence-based nucleosome ubiquitylation and binding assays.

a, Fluorescence-based nucleosome ubiquitylation assay. Left, ubiquitylation assays performed with nucleosomes labelled with Oregon Green 488 maleimide on H2A(Thr10Cys) mutant (H2A T10C-OG488). Right, replicates 1, 2 and 3 of wild-type assay run on four different gels and quantified to demonstrate reproducibility across gels. Data are mean and s.d., n = 4. b, Fluorescence-based nucleosome binding assay. Top left, nucleosomes labelled with Oregon Green 488 on H2B(Ser112Cys) mutant (H2B S112-OG488). Top right, binding of PRC1 ubiquitylation module leads to partial quenching of the fluorophore allowing affinity measurements to be made. Bottom, three technical replicates of fused wild-type PRC1 ubiquitylation module are shown to demonstrate reproducibility. Mean and s.d. shown for each data point, n = 3.

Extended Data Figure 7 Effects of PRC1 ubiquitylation module mutants on nucleosome ubiquitylation and binding.

a, Representative gel of one replicate of ubiquitylation assay using E1 Uba1, UbcH5c, STR-His10 tagged ubiquitin, NCPs, and E3 Ring1B–Bmi1 with the indicated Ring1B mutants stained with Coomassie (left) and scanned for fluorescent H2A (right). NCPs in the experiment are doped with NCPs containing the H2A(Thr10Cys) mutant labelled with Oregon Green 488 maleimide. b, Quantitation of mono-ubiquitylated (H2Aub1, dark blue), di-ubiquitylated (H2Aub2, blue) and tri-ubiquitylated (H2Aub3, light blue) H2A are shown as a fraction of total H2A. Data are mean and s.d. from three technical replicates. Samples from the same experiment were analysed on different gels, processed in parallel. c, Fluorescence quenching binding curves for fused PRC1 ubiquitylation modules containing the indicated mutations of Ring1B (coloured as shown). Mean and s.d. are shown for each data point, n = 3. Fluorescence is normalized to fit values for unbound and saturated NCPs. Concentrations depicted using log scale. dl, Experiments as described above for indicated Bmi1 mutants (d–f), UbcH5c charge reversal mutants (gi) and UbcH5c alanine mutants (jl). Triplicate ubiquitylation assays were repeated at least twice.

Extended Data Figure 8 Alignment of Bmi1 paralogues and E2s.

a, Sequence alignment of segments of Bmi1 with PCGF (Polycomb group RING finger) paralogues also found in PRC1. b, Sequence alignment of UbcH5c and other E2s known to be active (green) or inactive (red) or of unknown compatibility with Ring1B–Bmi1-mediated nucleosomal ubiquitylation. Key residues discussed in the text are indicated with an asterisk.

Extended Data Figure 9 Linker DNA increases the affinity of the PRC1 ubiquitylation modules for the nucleosome.

a, Linear B-form duplex DNA modelled onto the DNA ends of the PRC1 ubiquitylation module–NCP structure. b, The UbcH5c α4 helix occupies the major groove of modelled linker DNA. Several basic and aromatic side chains line the DNA-adjacent face of the α4 helix. c, Linker DNA enhances nucleosomal binding of fused PRC1 ubiquitylation module. Fluorescence quenching binding curves for the wild-type fused PRC1 ubiquitylation module binding to nucleosomes centred on 147-bp (black, grey and light grey) or 185-bp (blue and light blue) 601 DNA. Technical replicates performed on different days are depicted in different shades of grey and blue. Mean and s.d. are shown for each data point, n = 3. Fluorescence is normalized to fit values for unbound and saturated NCPs. Concentrations depicted using log scale.

Extended Data Figure 10 BRCA1 requires similar loop region and H2A–H2B acidic patch for nucleosome ubiquitylation.

a, Alignment of the BRCA1–BARD1 and Ring1B–Bmi1 heterodimers using the RING domains of BRCA1 from the BRCA1–BARD1 NMR structure35 (PDB code 1JM7) and Ring1B. BRCA1 and BARD1 are shown in orange and purple, respectively. Cα atoms of essential arginine residues are indicated by spheres. b, Sequence alignment of Ring1B and BRCA1 shows conserved nucleosome interacting loop. c, BRCA1(Lys70Ala/Arg71Ala) mutation eliminates E3 ligase activity of BRCA1–BARD1 RING heterodimer. Representative gel of one replicate of ubiquitylation assay using E1 Uba1, E2 UbcH5c, STR-His10 ubiquitin, NCPs, and E3 BRCA1–BARD1 with the indicated mutants stained with Coomassie and scanned for fluorescent H2A. NCPs in the experiment are doped with NCPs containing the H2A(Thr10Cys) mutant labelled with Oregon Green 488 maleimide. d, Quantification of mono-ubiquitylated (H2Aub1, dark orange), di-ubiquitylated (H2Aub2, orange) and tri-ubiquitylated (H2Aub3, light orange) H2A are shown as a fraction of total H2A. Mean and s.d. from three technical replicates are depicted. Ubiquitylation assay was repeated twice.

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McGinty, R., Henrici, R. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014). https://doi.org/10.1038/nature13890

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