1. Chromosomes and Gene Expression
  2. Structural Biology and Molecular Biophysics
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Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome

  1. Evan J Worden
  2. Xiangbin Zhang
  3. Cynthia Wolberger  Is a corresponding author
  1. Johns Hopkins University School of Medicine, United States
Research Article
Cite this article as: eLife 2020;9:e53199 doi: 10.7554/eLife.53199
6 figures, 2 tables, 2 data sets and 1 additional file

Figures

Figure 1 with 4 supplements
Architecture of the COMPASS H2B-Ub nucleosome complex.

(a) Cryo-EM structure of the COMPASS-nucleosome complex. The unsharpened EM density showing two COMPASS molecules bound to the nucleosome is depicted as a semitransparent surface. The sharpened EM density of the complex is depicted as an opaque surface and colored according to the different subunits of the complex. (b) The model of the COMPASS-nucleosome complex is shown and colored as in panel a. The unstructured histone H3 tail residues between the H3 exit point in the nucleosome and the Set1 active site are depicted as a blue dashed line. (c) Large-scale structural motions of COMPASS from the nucleosome-free state to the nucleosome-bound state. Free COMPASS (PDB: 6BX3) is colored gray and nucleosome-bound COMPASS is colored according to panel b. The largest motion in the structural transition at the end of Spp1 is shown as a black dashed arrow. (d) The COMPASS-nucleosome structure viewed from the dyad axis. The distances between the cis-H3 and trans-H3 subunits and the H3 residues in the Set1 active site are shown as dashed black lines.

Figure 1—figure supplement 1
Analysis of COMPASS activty and binding to the nucleosome.

(a) Electrophoretic mobility shift assay (EMSA) showing COMPASS binding to different Nucleosome (NCP) variants. (b) Quantification of the EMSA shown in panel a. The binding interaction between COMPASS and the H2B-Ub, H3K4Nle nucleosome is fit to single site binding equation. (c) Activity of COMPASS toward an isolated H3 peptide with and without SAM in the reaction.

Figure 1—figure supplement 2
Cryo-EM processing pipeline.
Figure 1—figure supplement 3
Cryo-EM map and model validation and example density.

(a) Fourier Shell Correlation (FSC) for the COMPASS-nucleosome structure. (b) Model-Map FSC curves calculated from the refined atomic coordinates and the full-map used for refinement (Full) and the half-maps used for validation (Half 1 and Half 2). (c) Local map resolution estimates calculated from ResMap (Kucukelbir et al., 2014) and colored according to local resolution. Top left, full sharpened map. Bottom left, a slice through the center of the map. (d) A vertical slice through the structure of COMPASS bound to the H2B-Ub nucleosome. All chains in the atomic model are shown as sticks and the EM density is shown as a gray mesh. (e) Example EM density around the H3 tail in the Set1 active site. (f) Example EM density for the SAM cofactor.

Figure 1—figure supplement 4
Detailed views of EM density in the structure.

In all panels the sharpened EM density is shown as a semi-transparent gray surface. (a) Superimposition of Spp1 from PDB: 6BX3 and Spp1 from this study. The fragmented EM density at the end of Spp1 corresponds N-terminal part of Spp1. (b) Detailed view of the contact between the Set1 RxxxRR helix and the H2A acidic patch. (c) Detailed view of the interaction between Set1 R936 and H2A D72. (d) Detailed view of the interaction between Set1 L928 and the Ubiquitin I36 patch. (e) Detailed view of the contact between Swd1 and the Ubiquitin I44 patch.

COMPASS makes multiple contacts with nucleosome DNA.

(a) Model of the COMPASS-nucleosome structure shown in cartoon representation and colored as in Figure 1. (b–d) Detailed views of COMPASS interactions with nucleosomal DNA. In all views the experimental EM density is shown as a semi-transparent gray surface. (b) DNA contact between Set1 and Bre2. Potential DNA-interacting COMPASS residues are shown in stick representation. (c) Contact between a flexible loop in Spp1 and the nucleosomal DNA. The unmodeled loop of Spp1 is depicted as a yellow dashed line and the positively charged, putative DNA-interacting residues in the loop are shown as yellow letters. (d) Interaction between Swd1 and the nucleosome DNA. Swd1 residues that contact DNA are shown in stick representation.

Figure 3 with 1 supplement
A conserved loop in Swd1 contacts the histone core.

(a) Close up view of the contact between Swd1 and the histone core. Swd1 is shown as a blue cartoon and the two loops that contact the histone core are colored red. (b) Detailed view of the specific contacts made between Loop 1 and Loop 2 of Swd1 and the histone core. Potential hydrogen bonds are shown as black dashed lines and the sharpened EM density is shown as a transparent gray surface. (c) Western blot analysis of H3K4 methylation states in swd1Δ yeast strains transformed with plasmids containing the indicated swd1 variants. (d) Quantification of the western blots from panel c. Error bars are the standard deviation of the data (n = 4).

Figure 3—source data 1

Western blot quantification for Swd1 Loop 1 mutants.

https://cdn.elifesciences.org/articles/53199/elife-53199-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Multiple sequence alignments of Swd1, Set1 and Bre2.

(a–c) Multiple sequence alignments were generated using the Clustle Omega software (Sievers et al., 2011). Species names are abbreviated (Sc = Saccharomyces cerevisiae, Sp = Schizosaccharomyces pombe, Kl = Kluyveromyces lactis, Dm = Drosophila melanogaster, Xt = Xenopus tropicalis, Mm = Mus musculus, Hs = Homo sapiens). Important features from the COMPASS structure are highlighted. (a) Multiple sequence alignment of Swd1 and its homologs. (b) Multiple sequence alignment of Set1 and its homologs. (c) Multiple sequence alignment of Bre2 and its homologs.

The RxxxRR helix binds to the H2A/H2B acidic patch.

(a) The COMPASS-nucleosome structure with COMPASS subunits surrounded by semi-transparent colored surfaces. The RxxxRR helix passes underneath the H2B-linked ubiquitin and is shown in cartoon representation. (b) Cutaway view of the COMPASS nucleosome structure showing the RxxxRR helix passing between COMPASS and the nucleosome. (c) Sharpened EM density surrounding the RxxxRR helix, which is shown in stick representation. (d–e) End-on view (d) and side view (e) of the RxxxRR helix. Contacting residues between the RxxxRR helix and the H2A/H2B acidic patch are shown in stick representation. (f) Detailed view of the section of Set1 that undergoes a conformational change. The corresponding part of Set1 in the isolated COMPASS structure (PBD: 6BX3, Apo) is colored yellow and shown as a cartoon. The Set1 residues that make contacts with the histone core and Ubiquitin are shown in stick representation and colored green. (g) Western blot analysis of H3K4 methylation states in set1Δ yeast strains transformed with plasmids containing the indicated set1 variants. (h) Quantification of the western blots from panel g. Error bars are the standard deviation of the data (n = 3).

Figure 4—source data 1

Western blot quantification for Set1 RxxxRR helix mutants.

https://cdn.elifesciences.org/articles/53199/elife-53199-fig4-data1-v2.xlsx
H2B-Ubiquitin recognition by different methyltransferases.

(a–c) Structures of H2B-ubiquitin activated methyltransferases bound to the H2B-Ub nucleosome. The structures are encompassed by a semitransparent gray surface, except for ubiquitin which is colored purple and depicted as a cartoon. Surface representations of the bound ubiquitin are shown on the right with the buried surface area colored red. The locations of the I44 and I36 patches are indicated with colored yellow and blue lines, respectively. (a) Structure of COMPASS bound the H2B-Ub nucleosome. (b) Structure of the MLL1 core complex bound to the H2B-Ub nucleosome (PDB: 6KIU). (c) Structure of Dot1L bound to the H2B-Ub nucleosome (PDB: 6NJ9). (d) Superimposition of the structures from a–c but only showing the ubiquitin to compare the relative positions of the H2B-linked Ubiquitin. H2B K120 is shown as spheres and colored blue.

Figure 6 with 1 supplement
Structural basis for COMPASS recognition of H2B-ub.

(a) Model of the COMPASS H2B-Ub nucleosome complex. The unsharpened EM density is shown as a semi-transparent gray surface. The connecting density between Bre2 and ubiquitin is shown. (b) Detailed view of the interactions made between Ubiquitin and Swd1 and Set1. Ubiquitin is enclosed in a semi-transparent pink surface and the interfacial residues are shown in stick representation. (c–d) Conformational changes in Swd1 that occur upon ubiquitin binding. The nucleosome-free (apo) structure of COMPASS is shown in gray (PDB: 6BX3). (c) Restructuring of the Swd1 C-terminal extension. (d) Restructuring of the Swd1 N-terminal extension. (e) Detailed view of Swd1 F9 interaction with ubiquitin. The sharpened EM density around Swd1 is shown as a semi-transparent gray surface and the EM density around ubiquitin is shown as a semi-transparent pink surface. (f) Detailed view of the interaction between Set1 L928 and ubiquitin. The sharpened EM density around Set1 is shown as a semi-transparent gray surface. (g) Western blot analysis of H3K4 methylation states in swd1Δ yeast strains transformed with plasmids containing the indicated swd1 variants. (h) Quantification of the western blots from panel g. Error bars are the standard deviation of the data (n = 4).

Figure 6—source data 1

Western blot quantification for Swd1 Ubiquitin-binding mutants.

https://cdn.elifesciences.org/articles/53199/elife-53199-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Comparison of Set1 catalytic domains from COMPASS in different states.

(a) Superimposition of the H2B-Ub nucleosome bound Set1 catalytic domain from S. cerevisiae (green) with the substrate bound, K. lactis Set1 catalytic domain from isolated COMPASS (PDB: 6CHG). (b) Superimposition of the H2B-Ub nucleosome bound Set1 catalytic domain from S. cerevisiae (green) with the substrate-free S. cerevisiae Set1 catalytic domain from isolated COMPASS (PDB: 6BX3).

Tables

Table 1
Cryo-EM data collection, refinement and validation statistics
Complex between COMPASS and the H2B-Ub nucleosome
(EMDB-21157)
(PDB 6VEN)
Data collection and processing
Magnification81,000
Voltage (kV)300
Electron exposure (e–/Å2)50
Defocus range (μm)−1.0 to −2.5
Pixel size (Å)1.08
Symmetry imposedC1
Initial particle images (no.)2,036,654
Final particle images (no.)179,588
Map resolution (Å)
FSC threshold
3.37 (0.143)
Map resolution range (Å)999–3.37
Refinement
Initial model used (PDB code)PDB: 6NJ9, 6BX3, 6CHG
Model resolution (Å)
FSC threshold
3.40 (0.5)
Model resolution range (Å)47.6 to 3.40
Map sharpening B factor (Å2)−122.6
Model composition
Non-hydrogen atoms
Protein residues
Ligands
24,032
2271
2
B factors (Å2)
Protein
Ligand
102.57
113.31
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.004
0.696
Validation
MolProbity score
Clashscore
Poor rotamers (%)
1.92
9.79
0.05
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)
94.01
5.99
0.0
Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Saccharomyces cerevisiae)Yeast strain BY4741(Baker Brachmann et al., 1998)
AntibodyAnti-Histone H3 (mono-methyl K4) polyclonalAbcamAbcam Cat# ab8895, RRID:AB_306847WB (1:500)
AntibodyAnti-Histone H3 (di-methyl K4) polyclonalAbcamAbcam Cat# ab7766, RRID:AB_2560996WB (1:500)
AntibodyAnti-Histone H3 (tri-methyl K4) polyclonalAbcamAbcam Cat# ab8580, RRID:AB_306649WB (1:500)
AntibodyAnti-GAPDH monoclonalAbcamAbcam Cat# ab125247, RRID:AB_11129118WB (1:2000)
Recombinant DNA reagentpEW106This StudypQE-81L: H3 K4M
Recombinant DNA reagentpEW66This StudyCOMPASS expression plasmid pBIG1a containing Bre2, Swd1, Swd2, Sdc1 and Sgh1
Recombinant DNA reagentpEW107This StudyCOMPASS expression plasmid pBIG1b containing 6xHis-3xFLAG-Set1(762–1080), Swd3 and twin-strep-Spp1.
Recombinant DNA reagentpEW108This StudyCOMPASS expression plasmid pBIG1ab containing all COMPASS subunits.
Recombinant DNA reagentWT Set1This StudypEW111 pRS415: WT Set1
Recombinant DNA reagentWT Swd1This StudypEW113
pRS415: WT Swd1
Recombinant DNA reagentSet1(R936A)This StudypEW118
pRS415: Set1(R936A)
Recombinant DNA reagentSet1(R936E)This StudypEW120
pRS415: Set1(R936E)
Recombinant DNA reagentSet1(R901A)This StudypEW123
pRS415: Set1(R901A)
Recombinant DNA reagentSet1(R904A)This StudypEW124
pRS415: Set1(R904A)
Recombinant DNA reagentSet1(R908A)This StudypEW125
pRS415: Set1(R908A)
Recombinant DNA reagentSet1(R909A)This StudypEW126
pRS415: Set1(R909A)
Recombinant DNA reagentSet1(R901A, R904A, R908A, R909A)This StudypEW127
pRS415: Set1(R901A, R904A, R908A, R909A)
Recombinant DNA reagentSet1(R901E)This StudypEW128
pRS415: Set1(R901E)
Recombinant DNA reagentSet1(R904E)This StudypEW129
pRS415: Set1(R904E)
Recombinant DNA reagentSet1(R908E)This StudypEW130
pRS415: Set1(R908E)
Recombinant DNA reagentSet1(R909E)This StudypEW131
pRS415: Set1(R909E)
Recombinant DNA reagentSet1(R901E, R904E, R908E, R909E)This StudypEW132
pRS415: Set1(R901E, R904E, R908E, R909E)
Recombinant DNA reagentSwd1(L12A)This StudypEW139
pRS415: Swd1(L12A)
Recombinant DNA reagentSwd1(E14R)This StudypEW140
pRS415: Swd1(E14R)
Recombinant DNA reagentSwd1(E14A)This StudypEW141
pRS415: Swd1(E14A)
Recombinant DNA reagentSwd1(F9A, L12A, E14A)This StudypEW142
pRS415: Swd1(F9A, L12A, E14A)
Recombinant DNA reagentSwd1(E397R)This StudypEW144
pRS415: Swd1(E397R)
Recombinant DNA reagentSwd1(V263A)This StudypEW145
pRS415: Swd1(V263A)
Recombinant DNA reagentSwd1(I264A)This StudypEW146
pRS415: Swd1(I264A)
Recombinant DNA reagentSwd1(N265A)This StudypEW147
pRS415: Swd1(N265A)
Recombinant DNA reagentSwd1(K266A)This StudypEW148
pRS415: Swd1(K266A)
Commercial assay or kitMTase-GloPromegaV7601

Data availability

Coordinates have been deposited in the PDB under accession code 6VEN. Maps have been deposited in EMDB under accession codes EMD21157.

The following data sets were generated
  1. 1
    RCSB Protein Data Bank
    1. EJ Worden
    2. C Wolberger
    (2020)
    ID 6VEN. Yeast COMPASS in complex with a ubiquitinated nucleosome.
  2. 2
    Electron Microscopy Data Bank
    1. EJ Worden
    2. C Wolberger
    (2020)
    ID EMD-21157. Yeast COMPASS in complex with a ubiquitinated nucleosome.

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