Abstract
The Rpd3S histone deacetylase complex has a crucial role in genomic integrity by deacetylating transcribed nucleosomes following RNA polymerase (Pol) II passage. Cryo-EM studies highlight the importance of asymmetrical Rco1–Eaf3 dimers in nucleosome binding, yet the interaction dynamics with nucleosomal substrates alongside elongating Pol II are poorly understood. Here we demonstrate the essential function of the Rco1 N-terminal intrinsically disordered region (IDR) in modulating Pol II association, in which K/R mutations within the Rco1 IDR impair interaction of Rpd3S with the C-terminal domain (CTD) of Rpb1, without affecting nucleosome recognition or complex integrity. We also identify the Rco1-PHD1 and Eaf3-CHD domains as crucial for specific binding to Ser5-phosphorylated CTD. The Rco1 IDR alleviates autoinhibition from its C terminus, facilitating PHD1-CHD engagement with phosphorylated CTD. Furthermore, we reveal a conserved mechanism by which asymmetrical Rco1–Eaf3 dimers coordinate nucleosome engagement and Pol II interaction, enhancing understanding of epigenetic complexes associated with transcriptional machinery.
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Data availability
Sequencing data have been deposited in the GEO database under the accession number GSE254580. Saccharomyces cerevisiae (S288C R64-2-1) and Schizosaccharomyces pombe (ASM294v2) genomes were used for genomic analysis. Published datasets from the PDB used in this study (7YI0, 7YI4 and 8I02) were further analyzed in Pymol (v.2.5.5). All unique materials used in this study are available from the lead contact with a completed Material Transfer Agreement. Source data are provided with this paper.
Code availability
The scripts used to analyze the data from this study are available via Zenodo at https://doi.org/10.5281/zenodo.14137822 (ref. 53).
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Acknowledgements
We are grateful to J. Liu, Y. Li, C. Wang and B. Cairns for S. pombe strain and culture. We also thank A. Karra for initially constructing engineered CTD plasmids. We would like to express our appreciation to the Experimental Nuclear Medicine Laboratory, Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine, for providing us with the experimental instruments. This work was supported by the National Key R&D Program of China (grant no. 2021YFA1300100 to B.L.), the National Natural Science Foundation of China (grants 32030019, 31872817 to B.L., 81901284 to Y.Y. and 32000413 to J.S.), the Youth Talent Support Program of the College of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine (grant 2024RCZC-C-01 to Y.P.) and the Fundamental Research Funds for the Central Universities.
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B.L., Y.L, W.W. and Y.P. conceptualized the study. Y.P., C.R. and Y.N. performed biochemistry, genomics and yeast in vivo phenotype experiments, with the help from M.H., N.L. and M.H. Y.P. reconstituted Rpd3S with help from M.P., H.G. and P.W. M.L. performed S. pombe manipulation and genomics. Y.P. performed the in vitro phase separation assay, with the help from J.X. and N.L. Y.P. and Q.Z. performed bioinformatics analyses, with the help from J.S. B.L. and Y.P. wrote the manuscript. B.L. and H.L. supervised the project. B.L., Y.Y., J.S. and Y.P. acquired funding.
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Extended data
Extended Data Fig. 1 Rco1-B of Rpd3S undergoes dramatic conformational changes after binding to nucleosomes.
Structural comparison of the apo and nucleosome-bound states of Rpd3S. The upper and lower panels show two views of the apo-Rpd3S complex (PDB: 7YI0) and nucleosome-bound Rpd3S (PDB: 7YI4) respectively. The color scheme of each Rpd3S subunit is indicated at the top along the domain structure illustration of Rco1 and Eaf3.
Extended Data Fig. 2 Rco1 K/R mutations impair the in vivo functions of Rpd3S.
a, Construction strategy for Rco1 mutant strains utilizing the delitto perfetto system involved inserting the CORE cassette (blue box) with a KlURA marker at the 150th amino acid position of RCO1. Subsequently, this cassette was replaced by DNA fragments encoding N-terminally FLAG-tagged Rco1 mutants through the induction of double-strand breaks. b, An illustration depicting codon-optimized Rco1 mutants, with regions highlighted in purple indicating codon optimization based on S. cerevisiae preferences. c, Western blot analysis of fractionated cytoplasmic and nuclear extracts from yeast cells expressing FLAG-tagged Rco1 wild-type (WT) or mutant variants. Taf5 and Taf6 were used as markers for the nuclear fraction, while Pgk1 served as a cytoplasmic marker to confirm successful cellular fractionation. ‘Total’ denotes lysates prior to fractionation, ‘CE’ represents cytoplasmic extracts, and ‘NE’ indicates nuclear extracts. d, Pearson correlation analysis was performed on H4 acetylation ChIP-seq experiments, demonstrating high reproducibility between two biological replicates. The Pearson correlation coefficient was calculated using multiBamSummary, considering the read coverage of 1 kb-binned matrices.
Extended Data Fig. 3 The phase separation property of IDR is determined by the presence of a K/R-enriched region.
a, Recombinant WT and mutant Rco1 IDR N (1-260) constructs were depicted, featuring an N-terminal GST-tag followed by a TEV protease cleavage site. b, Purified GST-WT and mutant Rco1 IDRs were visualized using Coomassie blue staining. c, Droplet formation assays demonstrated the phase separation of GST-Rco1-N (1-260)-EGFP at various protein concentrations supplemented with 20% PEG-8000 crowding agent. d, Droplet formation of GST-Rco1-N (1-260)-EGFP was observed at different NaCl concentrations. e, A schematic of the droplet sedimentation assay was provided. f, Coomassie blue staining was performed on samples from droplet sedimentation assays of GST-Rco1-N (1-260)-EGFP with varying dextran concentrations, including the input (InP), supernatant (S), and pellet (P) fractions. g, Quantification of GST-Rco1-N (1-260)-EGFP sedimentation assay from (f) was conducted based on 3 biological replicates. Data are presented as mean ± SD. h, Droplet formation assay of WT, 13 K/R > A, and 31S > A mutants of GST-Rco1-N (1-260)-EGFP was performed. i, Droplet size in (h) was quantified using OpenCFU software, considering droplets between 100 and 500 pixels only. n = 796 and 565 from three independent experiments. Error bars represent the median with interquartile range. j, Coomassie blue staining was conducted on samples from the sedimentation assays of WT and mutant GST-Rco1-N (1-260)-EGFP. k, Quantification of sedimentation assays of GST-Rco1-N (1-260)-EGFP from (j) was presented based on 3 biological replicates, accompanied by P values obtained from a two-sided Student’s t-test. Data are presented as mean ± SD.
Extended Data Fig. 4 Analysis of CTD Interactors Using GST-CTD Pull-down Assay.
a, Coomassie blue staining shows purified WT and mutant Rpd3S complex from insect cells. b, Engineered CTD variant WT28 with modifiable sequences retains full functionality, illustrated by amino acid alignment and labeled non-consensus residues and structural regions. c, Diagram of ‘carriers’ vector for transporting engineered CTD quarters. d, Assembly of ‘mothership’ CTD involves subcloning into downstream vectors, with schematic of GST-CTD28 and its S2,5 A mutant. e, Plasmid shuffle assay tests functionality of CTD_WT, CTD28, and S2,5 A mutant in Rpb1, using pRS415 as control. f, Western blots assess phosphorylation of S5P, S2P, and S7P in GST-CTD28 by specific kinases. g, Flowchart details the GST-CTD28 pull-down assay process. h, Pull-down assays explore Set2 binding at varying NaCl concentrations, post-phosphorylation by BUR or CTDK-I kinase. i, Coomassie blue staining of WT and mutant Rpd3S complexes from insect cells, noting non-specific bands. j, GST-CTD_WT pull-down assays on WT and mutant Rpd3S complexes with FLAG-Sin3 purified from insect cells.
Extended Data Fig. 5 One Arm of Rpd3S controls its CTD binding.
a, Coomassie blue staining displays the Rpd3S complex resulting from single V5 purification or V5/HA tandem purification. b-e, GST-CTD28 pull-down assays were conducted to assess the CTD binding affinity of Rpd3S complexes. b, FLAG/HA tandem purified Rpd3S complexes were used in the assay under 100 mM NaCl. c, Quantification of the western blot results from (b) based on four independent experiments. Data are presented as mean ± SD. d, The GST-CTD pull-down assay was performed using V5/HA tandem purified Rpd3S complexes under 100 mM NaCl. e, Quantification analysis of the western blot results depicted in (d). f, Coomassie blue staining of Rpd3S complexes purified through single FLAG or FLAG/HA tandem purification. An asterisk indicates a non-specific band. g, GST-CTD28 pull-down assays were carried out to examine the CTD binding affinity of Rpd3S variants. V5/HA tandem purified Rpd3S complexes were used in the assay under 100 mM NaCl. h, Quantification of the western blot results from (g) based on two independent experiments.
Extended Data Fig. 6 The Rpd3S complexes in both S. pombe and S. cerevisiae exhibit evolutionarily conserved functional activities.
a, Pearson correlation analysis was performed on S. pombe H4 acetylation ChIP-seq experiments, demonstrating high reproducibility between two biological replicates. MultiBamSummary was used to calculate the read coverage of bin-size 1 kb. b, Principal component analysis (PCA) was conducted on RNA-seq data from two replicates of WT, cph1Δ, cph2Δ, and set2Δ strains. WT1 is isogenic to cph1Δ and cph2Δ, while wt2 is isogenic to set2Δ. c, spRpd3S was purified from S. pombe yeast strain using Pst2-FLAG tag, followed by silver staining. d, Coomassie blue staining was performed on HIS-tag purified HIS-PHD1-MID/GST-Alp13 heterodimers. e, A schematic was provided to illustrate the process of obtaining GST-cleaved HIS-PHD1-MID/Alp13 dimers through HIS-tag purification. f, Ponceau S staining was conducted on GST-digested HIS-PHD1-MID/Alp13. g, A schematic representation of Cph2 mutant constructs was shown, indicating predicted disordered regions based on the PONDR-VSL2 algorithm. Red lines represent K/R to A mutations. h, Sedimentation assay of WT and K/R > A mutants of GST-Cph2 N (1-260)-EGFP revealed that K/R > A mutants of Cph2 inhibit phase separation. i, Quantification analysis of the sedimentation assay in (h) is presented based on 3 biological replicates. Data are presented as mean ± SD.
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Pan, Y., Liu, M., Ruan, C. et al. Inherent asymmetry of Rpd3S coordinates its nucleosome engagement and association with elongating RNA polymerase II. Nat Struct Mol Biol 32, 687–697 (2025). https://doi.org/10.1038/s41594-024-01453-w
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DOI: https://doi.org/10.1038/s41594-024-01453-w