Abstract
Crosstalk between histone modifications represents a fundamental epigenetic mechanism in gene regulation. During the transcription elongation process, the histone deacetylase complex Rpd3S is recruited to H3K36-methylated nucleosomes to suppress cryptic transcription initiation. However, how subunits of Rpd3S are assembled and coordinated to recognize nucleosomal substrates and exert their deacetylation function remains unclear. Here we report the structure of Saccharomyces cerevisiae Rpd3S deacetylase bound to H3K36me3-modified nucleosome at 3.1 Å resolution. It shows that Sin3 and Rco1 subunits orchestrate the assembly of the complex and mediate its contact with nucleosome at multiple sites, with the Sin3–DNA interface as a pivotal anchor. The PHD1 domain of Rco1 recognizes the unmodified H3K4 and places the following H3 tail toward the active site of Rpd3, while the chromodomain of Eaf3 subunit recognizes the H3K36me3 mark and contacts both nucleosomal and linker DNA. The second copy of Eaf3-Rco1 is involved in neighboring nucleosome binding. Our work unravels the structural basis of chromatin targeting and deacetylation by the Rpd3S complex.
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Data availability
The electron density reconstructions and final models were deposited with the Electron Microscopy Data Bank (EMDB; accession codes EMD-35081, 35082, 35083, 35084 and 36283 for Rpd3S, Rpd3S–nucleosome, Eaf3–nucleosome, Rpd3S–nucleosome composite map and Rpd3S–di-nucleosome, respectively) and with the Protein Data Bank (PDB; accession codes 8HXX, 8HXY, 8HXZ, 8HX0 and 8JHO, respectively). Source data are provided with this paper.
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Acknowledgements
We thank S. Chang and X. Zhang from the Centre of Cryo-Electron Microscopy at Zhejiang University for maintaining the facility and assistance with data collection. We thank C. Bi from the Core Facilities, Zhejiang University School of Medicine for her technical support. We thank K. Cheng from Hangzhou Normal University for proofreading of the manuscript. H.W. was supported by National Natural Science Foundation of China (grants 32370611 and 22DAA00673) and the Start-up Funding from the Second Affiliated Hospital, Zhejiang University School of Medicine (grants Y1103372023 and 1942222R3/058). Z.L. was supported by the National Natural Science Foundation of China (grants 82188102 and 82030074).
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H.W. conceived the project, supervised the research and carried out all experiments and data analysis unless stated otherwise. W.L. performed the yeast strain construction and western blot. H.C. was involved in part of model building and structure refinement under the supervision of H.W. Z.L. contributed to experiment design. H.W. evaluated and interpreted the data. H.W. and H.C. wrote the manuscript with the input from all authors.
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Extended data
Extended Data Fig. 1 Cryo-EM structure determination of Rpd3S-nucleosome complex.
a, SDS–PAGE of peak fraction of Rpd3S in complex with di-nucleosome used for cryo-EM grid preparation. Experiments were performed one time. For gel source data, see Supplementary Fig. 1. b, A representative cryo-EM micrograph showing particles of Rpd3S-nucleosome complex. Experiments were performed at least three times. c, Representative 2D class averages of the complex particles exhibiting major conformational heterogeneity. d, Sorting and classification tree used to reconstruct Rpd3S-di-nucleosome complex. e, Sorting and classification tree used to reconstruct Rpd3S-mono-nucleosome complex. Masks applied during classification and refinement are depicted as translucent outlines and specified in the accompanying text in blue. Complex subunits and submodules are highlighted according to the color codes in Fig. 1. Final reconstructions that were deposited to the EMDB are boxed with a light grey background and outlined in black.
Extended Data Fig. 2 Quality of cryo-EM reconstructions.
a, Local resolution estimation of three determined complexes, including Rpd3S-nucleosome (left), Rpd3S (middle) and Eaf3CHD-nucleosome (right). The color bars from blue to red imply the local resolution range (Å). The angular distribution diagrams of particles in the final reconstructions are shown on the bottom right with color shading from blue to yellow being related to particle numbers at specific orientations. b, Fourier shell correlation (FSC) between the half maps of each reconstruction. The average resolution is estimated at the FSC 0.143 cut-off criterion (dashed line). c, Densities for various parts of Rpd3S-nucleosome complex as indicated. d, Model-to-map FSCs between the final models and their respective reconstructions. The resolutions at the FSC 0.5 cut-off criterion (dashed line) are denoted.
Extended Data Fig. 3 Subunit domain structures and their inter-molecular interactions.
a–b, Domain arrangements of Rco1L and Sin3 in the Rpd3S complex. c, Interactions between Sin3 HID C-module (ribbon model) and Rpd3 (electrostatic surface). d, Interactions between Sin3 HID C-module (electrostatic surface) and Rco1L N-terminal domain (ribbon model). Interacting residues of Rco1L are indicated as sticks. e, Superimposition of Sin3 HID C-module structure with the previously published Sin3A HID structure (PDB code 2N2H)17. f, Detailed interactions between Sin3 HID N-module and Eaf3 MRG. Residues involved in interactions are illustrated as sticks. Yellow dashed lines represent polar interactions. g, Detailed interactions between Rpd3 and Sin3 HID N-module. Residues mediating subunit contacts are represented as sticks. h, Superimposition of Sin3 PAH3 and Sin3A PAH3 (PDB code 2LD7)18. i, Interactions between Sin3 and Rco1L PHD2. Highlighted as sticks are residues involved in the interactions between the two proteins. Yellow dashed lines denote hydrogen bonds. This view was obtained by rotating Fig. 2b counter clockwise 145° around Y-axis. j, Detailed interactions between Rco1L PHD2 and Rpd3 with associated residues represented as sticks. Yellow dashed lines stand for polar interactions.
Extended Data Fig. 4 Detailed interactions between the MRG domain of Eaf3 and Rco1L.
a–c, Detailed molecular interactions between Eaf3 MRG and Rco1L PHD1-SID. Residues involved in inter-molecular interactions are displayed as sticks. Yellow dashed lines denote polar interactions.
Extended Data Fig. 5 Structural comparison between Rpd3 and HDACs.
a and b, Superimposition of Rpd3 structure with the structure of HDAC3 (PDB code 4A69)19 in (a) or HDAC8 (PDB code 1W22)20 in (b). c and d, Side by side structural comparison between Rpd3 and HDAC8 (PDB code 1W22)20 reveals a similar active site configuration. Yellow dashed lines represent polar interactions. The inhibitor show in d is N-hydroxy-4-{methyl[(5-pyridin-2-yl-2-thienyl)sulfonyl]amino}benzamide. e and f, Side by side structural comparison of the activation mechanism between Rpd3 and HDAC3 bound to the deacetylase activation domain (DAD) from the human SMRT (the silencing-mediator of retinoid and thyroid) co-repressor (PDB code 4A69)19. Ins(4,4,5,6)P4 in f stands for D-myo-inositol-(1,4,5,6)-tetrakisphosphate.
Extended Data Fig. 6 Histone tails deacetylated by Rpd3S.
a, The density of histone peptide bound to Rpd3S. Two possible histone H3 tail fragments could be assigned to this density. H3 residues 8–20 was assigned according to the continuity of the density from the H3 N-terminus bound to the Rco1 PHD1 towards the active site of Rpd3 (upper). Alternatively, H3 residues 13–25 was assigned based on the register of bulky side chains (low). b, H3K9 is unreachable to the active site of Rpd3. The distance from K9 residue to the active site of Rpd3 is 15 Å which is too large for the coiled linker (T6-R8) to span. c, The H4 tail is close to the Rpd3S active site. The N-terminal tail of H4 is just beneath the active site of Rpd3S with a distance of 30 Å from the resolved terminal residue of H4 to the lysine in the catalytic pocket in the structure. d, Rpd3S binds to the nucleosome to release the unspecific attachment of H4. The H4 tail may attach to nucleosomal DNA. Binding of Rpd3S at SHL –2 may drive the H4 towards the active site.
Extended Data Fig. 7 Effects of disruption of interactions between Rpd3S subunits and DNA.
a, Serial dilution assays of each mutant strain. Mutations in each strain are list in the table below. Plates were scanned with a black background. Experiments were performed three times. b, Histone acetylation level detection by Western blot on each site. Antibodies used against acetylation on each site were indicated on the left. Moderate increase of H3K9ac level in Sin3 mutant strain may come from the impaired function of Rpd3L complex which also contains the Sin3 subunit and deacetylates nucleosomes at promoter regions. Experiments were performed three times.
Extended Data Fig. 8 Structure of the Rpd3S-di-nucleosome complex.
a, Model fitted into the Rpd3–di-nucleosome complex reconstruction. Low-threshold contoured map of the complex is displayed as semitransparent surface. A model of Rpd3S bound to di-nucleosome was nicely fitted into the density. b, The second copy of the Eaf3-Rco1 module might bind the N-terminal tail of histone H3 from a neighbouring nucleosome. Focused refinement map of Eaf3-Rco1in Rpd3S–mono-nucleosome reconstruction shown at low threshold revealed a weak density could be assign to the histone H3 N-terminus from the neighbouring nucleosome at the binding surface of the second Rco1 PHD1.
Supplementary information
Supplementary Video 1
The video shows an overview of Rpd3S–nucleosome complex structure. It first depicts the overall shape of the Rpd3S–nucleosome based on a composite cryo-EM density map. It then shows the architecture of Rpd3S and interfaces between subunits. Finally, it shows the interactions of Rpd3S with the H3K36me3-containing nucleosome.
Supplementary Video 2
The video shows different modes of relative movement of Rpd3S around the nucleosome.
Supplementary Video 3
The video shows how two copies of the Rco1-Eaf3 histone recognition submodule in Rpd3S bind to the di-nucleosome substrate.
Source data
Source Data Fig. 3, Extended Data Figs. 1 and 7.
This figure denotes the uncropped version of the western blots, gel and plate images shown in Fig. 3d and Extended Data Figs. 1a and 7. The black boxes highlight the cropped areas.
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Li, W., Cui, H., Lu, Z. et al. Structure of histone deacetylase complex Rpd3S bound to nucleosome. Nat Struct Mol Biol 30, 1893–1901 (2023). https://doi.org/10.1038/s41594-023-01121-5
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DOI: https://doi.org/10.1038/s41594-023-01121-5
