Binding to nucleosome poises human SIRT6 for histone H3 deacetylation
- Department of Integrated Structural Biology, IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), France
- Université de Strasbourg, IGBMC UMR 7104-UMR-S 1258, France
- CNRS, UMR 7104, France
- Inserm, UMR-S 1258, France
- Université de Lorraine and CNRS, UMR 7019 LPCT, France
Figures
Figure 1 with 5 supplements
Structure of human SIRT6 in complex with the nucleosome.
(a) Front (top) and side (bottom) views of a composite cryo-EM reconstruction of human SIRT6-nucleosome. Maps from focused refinements of SIRT6 (magenta – Rossmann fold and Zn-finger domains …
Figure 1—figure supplement 1
SIRT6 lacks the helix bundle between the Rossmann fold and the Zinc-finger domains.
The structures of human sirtuins are depicted – the Rossmann fold domain in blue, Zinc-finger in tan and the helix bundle between the two in green.
Figure 1—figure supplement 2
Cryo-EM data analysis strategy for SIRT6-nucleosome – dataset 1.
(a) Complete data processing scheme. Maps colored in rainbow represent the local resolution of the reconstructions. Hollow blue volume represents the mask used for focused classification and …
Figure 1—figure supplement 3
Cryo-EM data analysis strategy for SIRT6-nucleosome – dataset 2.
Complete data processing scheme. Maps colored in rainbow represent the local resolution of the reconstructions. FSC curves are depicted as a function of resolution in angstrom. CryoSPARC v.3 and v.4 …
Figure 1—figure supplement 4
Representative regions illustrating the quality of the cryo-EM map of SIRT6 bound to nucleosome.
(a) Close-up view of the SIRT6 Zn-finger interacting with the nucleosomal acidic patch. (b) View showing map details around the nucleosome dyad. (c) SIRT6 Rossmann fold domain interaction with the …
Figure 1—figure supplement 5
SIRT6 binds to and displaces the “looser” DNA terminus.
(a) Cryo-EM densities of the nucleotide base pairs at the dyad (yellow) and positions ± 4 (green) showing the orientation of the DNA in the SIRT6-nucleosome complex. Besides the dyad, the positions …
Figure 2 with 2 supplements
Flexibility of the SIRT6 Rossmann fold.
(a) UMAP projection of the latent embeddings of a subset of SIRT6-nucleosome particles representing the concerted movement of the Rossmann fold and the DNA terminus. (b) Structural representation of …
Figure 2—figure supplement 1
Schematic representation of CryoDRGN analyses.
Particle images from CryoSPARC refinement were Fourier cropped to a size of 128x128 pixels. They were subjected to CryoDRGN analysis (run1). Ten representative volumes of the first Principal …
Figure 2—figure supplement 2
SIRT6 binding renders DNA termini more flexible.
(a) Projection of per residue flexibility contribution calculated from molecular dynamics simulations of the nucleosome without SIRT6. The balls at the DNA residues are colored according to the …
Figure 3 with 3 supplements
Binding of SIRT6 to the nucleosome.
(a) Close-up view on zinc-finger interactions with the acidic patch. Color code as in Figure 1. (b) Protein sequence alignment of human sirtuins. Red boxes and asterisks depict the residues of SIRT6 …
Figure 3—figure supplement 1
WT and mutant SIRT6 - interactions with nucleosome and deacetylation activity.
(a) Electrophoretic mobility shift assay to test binding of Widom-601 nucleosome to wt and mutant SIRT6. Lower and upper shifted bands visible in SIRT6 wt lane likely correspond to one or two SIRT6 …
Figure 3—figure supplement 2
SIRT6 rearrangement upon nucleosome binding.
(a) Top view of the SIRT6 bound nucleosome. Two distinct densities were observed between SIRT6 and the DNA. (b) Side view of the SIRT6 bound nucleosome. The third helix of SIRT6 (α3), which is part …
Figure 3—figure supplement 3
Electrophoretic mobility shift assay comparing SIRT6 binding to H2A.
Z containing nucleosomes and canonical (H2A containing) ones. Used for creating Figure 3f.
Figure 4
SIRT6 poised to deacetylate lysine residues of H3.
(a) Side View of SIRT6 bound to the nucleosome with histone H3 K9 residue (red sphere) closest to the SIRT6 active site (blue spheres) from a set of molecular dynamics simulations. (b) Side View of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21-RIL | N/A | ||
| Recombinant DNA reagent | SIRT6 (plasmid) | AddGene | #28271 | SIRT6 (PDB: 3PKJ) in pET28a-LIC |
| Recombinant DNA reagent | Mutant SIRT6 (DNA fragment) | SynBio | K170A, R172A, R175A, R178A, R220A, R231A and R248A | |
| Recombinant DNA reagent | Widom601 DNA (plasmid) | Dyer et al., 2004 | Gift from Dr. K. Mohideen-Abdul. | |
| Recombinant DNA reagent | X. laevis H2A, H2B, H3, H4 histones (plasmids) | Luger et al., 1999; Dyer et al., 2004 | Gift from Dr. K. Mohideen-Abdul. | |
| Recombinant DNA reagent | Human H2A.Z.1 (plasmid) | AddGene | #42629 | H2A.Z.1 in pET28a |
| Antibody | H3K27ac (rabbit monoclonal) | CellSignaling | #8173 S | Diluted: 1:1000 |
| Antibody | H3K9ac (rabbit polyclonal) | Abcam | Ab4441 | Diluted: 1:2500 |
| Antibody | H3 (mouse monoclonal) | CellSignaling | #14269 S | Diluted: 1:1000 |
| Software, algorithm | SerialEM | v4.1 | Automated data collection | |
| Software, algorithm | EPU | Thermo Fisher | v3.4 | Automated data collection |
| Software, algorithm | Warp | Warp | v1.0.9 | Micrograph preprocessing |
| Software, algorithm | cryoSPARC | cryoSPARC | v4 | Image analysis |
| Software, algorithm | RELION | RELION | v3.1 | Image analysis |
| Software, algorithm | crYOLO | crYOLO | v1.9.6 | Particle picking |
| Software, algorithm | cryoDRGN | cryoDRGN | v1.1 | Image analysis – structural flexibility analysis |
| Software, algorithm | DeepEMhancer | DeepEMhancer | v0.8 | Map sharpening |
| Software, algorithm | UCSF Chimera/ChimeraX | UCSF ChimeraX | v1.16/1.6 | Rigid body fitting, visualisation and figures |
| Software, algorithm | Isolde | Isolde | v1.6 | Flexible fitting, model refinement |
| Software, algorithm | PHENIX | PHENIX | v1.21 | Model refinement |
| Software, algorithm | AMBER20 suit | AMBER20 | MD simulations |
Additional files
-
Supplementary file 1
Supplementary information for cryo-EM and molecular dynamics symulations.
(a) Cryo-EM data collection, refinement and validation statistics. (b) Frequency of conformational clusters for each MD ensemble. Clusters featuring the H3 tail protruding between the DNA and the octamer are marked by a star.
- https://cdn.elifesciences.org/articles/87989/elife-87989-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/87989/elife-87989-mdarchecklist1-v1.docx
