Abstract
Cells reprogram their transcriptomes to adapt to external conditions. The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a highly conserved transcriptional coactivator that plays essential roles in cell growth and development, in part by acetylating histones. Here, we uncover an autoregulatory mechanism of the Saccharomyces cerevisiae SAGA complex in response to environmental changes. Specifically, the SAGA complex acetylates its Ada3 subunit at three sites (lysines 8, 14 and 182) that are dynamically deacetylated by Rpd3. The acetylated Ada3 lysine residues are bound by bromodomains within SAGA subunits Gcn5 and Spt7 that synergistically facilitate formation of SAGA homo-dimers. Ada3-mediated dimerization is enhanced when cells are grown under sucrose or under phosphate-starvation conditions. Once dimerized, SAGA efficiently acetylates nucleosomes, promotes gene transcription and enhances cell resistance to stress. Collectively, our work reveals a mechanism for regulation of SAGA structure and activity and provides insights into how cells adapt to environmental conditions.
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Data availability
Uncropped blots and source data are provided with this paper. The RNA-seq and ChIP–seq for RNA pol II data reported in this paper have been deposited in the NCBI with the accession numbers GSE161887 and PRJNA764426. Source data are provided with this paper.
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Acknowledgements
We especially thank J. L. Workman (Stowers Institute for Medical Research) for reagents and suggestions on this project. We sincerely thank H. Li (Tsinghua University) and S. Wu (Hubei University) for assistance on SAGA purification. We thank Z. Wang and J. Yu (Wuhan University) for assistance in glycerol density gradient centrifugation. We thank D. Wei (Huazhong Agricultural University) for assistance in molecular dynamic simulations. This work was supported by funding from National Natural Science Foundation of China (31872812 to X. Y., 31970578 to S. L.), Natural Science Foundation of Hubei Province (2019CFA077 to X. Y., 2021CFA013 to S. L.).
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X. Y. and S. L. conceptualized the study. Experiments were performed by J. Huang, W. D. and D. X. Mass spectrometry was performed and results were analyzed by J. Huang, Q. X. and F. G. Glycerol density gradient centrifugation was performed by J. Huang and W. D. Mononucleosomes and dinucleosomes were prepared by C. L. and J. Hu. The original draft was written by X. Y. and S. L. Reviewing and editing of the manuscript was done by X. Y. and S. L. X. Y. and S. L. supervised the work.
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Extended data
Extended Data Fig. 1 SAGA acetylates Ada3 at lysines 8, 14 and 182.
a, 5 μg purified Ada2/Gcn5/Ada3 subcomplex was incubated with 0.5 mM acetyl-CoA in 40 μl HAT buffer for 30 min. The reaction components were resolved on a 15% SDS-polyacrylamide gel followed by western blots with pan acetyl-lysine antibody. b-d, Analysis of the acetylation sites on Ada3 by mass spectrometry. e, Western blot analysis of Ada3 and Gcn5 in WT, Ada3-K8R, Ada3-K14R, Ada3-K182R, Ada3-K8R/K14R, Ada3-K8R/K182R, Ada3-K14R/K182R and Ada3-3KR when grown in YP + 2% glucose (YPD). f, Dot blot assay of the specificity of anti-Ada3K14ac and anti-Ada3K182ac antibodies. The Ada3 peptides containing acetylated or unacetylated lysine 14, or lysine 182 were immunoblotted with anti-Ada3K14ac and anti-Ada3K182ac antibody, respectively. Different dots represent different diluted peptides as indicated. g, Western blot analysis of cell lysates of WT, Ada3-K14R, and Ada3-K182R with anti-Ada3K14ac and anti-Ada3K182ac antibody, respectively. h, Ada3 acetylation was unaffected by FLAG tagging. i, Analysis of Ada3 acetylation in deletion mutants of different SAGA subunits by western blots. j, Silver staining of purified Wt SAGA and SAGA Ada3-3KR. k, Ada3 was acetylated by SAGA complex at lysines 8, 14 and 182 in vitro. 20 ng purified Wt SAGA and SAGA Ada3-3KR were incubated with 0.5 mM acetyl-CoA at 30 °C for 0-30 min. For Extended Data Fig. 1e-j, shown is the typical example of two biological independent experiments. For Extended Data Fig. 1k, shown is the typical example of three biological independent experiments.
Extended Data Fig. 2 Ada3 is dynamically acetylated within SAGA complex.
a, Silver staining of SAGA purified from cells grown in glucose-containing (Glu) or sucrose-containing (Suc) medium. b, Ada3 acetylation was specifically induced by sucrose. SAGA was TAP purified from cells grown in glucose-containing (Glu) or sucrose-containing (Suc) medium. Acetylated subunits were determined by pan acetyl-lysine antibody. c, Ada3 acetylation was induced when grown in phosphate starvation media. WT, gcn5Δ, and Ada3-3KR cells were grown in YPD overnight and then switch to SC plus 1g/liter KH2PO4 phosphate (SC + Pi) medium or SC medium plus 1g/liter KCl (SC - Pi) for 3 hrs. d, Analysis of Ada3 acetylation in deletion mutants of histone deacetylases. e, Rpd3 deacetylates Ada3 in vitro. Purified SAGA was incubated with 0.5 mM acetyl-CoA (AcCoA) for 1 hr and then immobilized on calmodulin beads to remove excess acetyl-CoA. 20 ng purified Rpd3 complex was then added and the reaction was performed at 30 °C for 0-1 hr. The HDAC inhibitor, TSA was added as a negative control. The bead-bound proteins were boiled and Ada3 acetylation was analyzed by immunoblots. f, The interaction between Rpd3 and SAGA was not primarily affected by Ubp8. Rpd3-FLAG was immunoprecipitated from Rpd3-FLAG and Rpd3-FLAG ubp8Δ mutant with anti-FLAG. g, Analysis of Ada3 acetylation in WT, rpd3Δ, and Ada3-3KR rpd3Δ cells grown in SC + Pi or SC – Pi medium. h, Western blot analysis showing sucrose treatment did not change Rpd3 expression. i, Silver staining of Rpd3 complex purified from cells grown in glucose-containing (Glu) or sucrose-containing (Suc) medium. For Extended Data Fig. 2d, h, data represent means ± SE; n=3 biological independent experiments; two-sided t-tests were used for statistical analysis. For Extended Data Fig. 2a-c, e-g, i, shown is the typical example of at least two biological independent experiments.
Extended Data Fig. 3 SAGA undergoes Ada3 acetylation-dependent dimerization.
a, b, Glycerol density gradient centrifugation analysis of purified Wt SAGA incubated without (a) or with (b) acetyl-CoA. 50 µg Wt SAGA was incubated with 10 mM acetyl-CoA at 30 °C for 15 min. The reaction products were applied to a 10-50% glycerol density gradient centrifugation. The fractions were analyzed by Western blots with indicated antibodies and silver staining. c, d, Glycerol density gradient centrifugation analysis of SAGA Ada3-3KR treated without (c) or with (d) 10 mM acetyl-CoA. e, Silver staining of purified SAGA monomer and SAGA dimer. The fractions 11-15 in Fig. 3a were pooled and concentrated as SAGA monomer. The fractions 19-23 in Fig. 3b were pooled and concentrated as SAGA dimer. f, Quantification of fractions in Figs. 3a-3d. g, In vivo Co-IP showed that Ada3 acetylation is required for interaction between Ada3-FLAG-containing SAGA and Ada3-Myc-containing SAGA. Ada3-FLAG was immunoprecipitated from cell extract of the diploid strain Ada3-FLAG/Ada3-Myc, Ada3-3KR-FLAG/Ada3-Myc, Ada3-FLAG/Ada3-3KR-Myc, and Ada3-3KR-FLAG/Ada3-3KR-Myc, respectively. The co-IPed Ada3-Myc was detected by anti-Myc. Compared with the interaction between SAGA Ada3-FLAG and SAGA Ada3-Myc, the interaction between SAGA Ada3-3KR-Flag and SAGA Ada3-Myc as well as the interaction between SAGA Ada3-Flag and SAGA Ada3-3KR-Myc were reduced. No obvious interaction was detected between SAGA Ada3-3KR-FLAG and SAGA Ada3-3KR-Myc. h-k, In vivo Co-IP showing the self-association of SAGA complex. For Extended Data Fig. 3a-e, g, shown is the typical example of two biological independent experiments. For Extended Data Fig. 3h-k, shown is the typical example of two biological independent experiments.
Extended Data Fig. 4 Ada3 acetylation-dependent SAGA dimerization is enhanced when grown in sucrose or phosphate starvation conditions.
a, b, Glycerol density gradient centrifugation analysis of Wt SAGA (a) and SAGA Ada3-3KR (b) purified from cells grown in YP + 2% glucose or YP + 2% sucrose. c, Glycerol density gradient centrifugation analysis of SAGA rpd3Δ purified when cells were grown in YP + 2% glucose. d, Quantification of fractions in Extended Data Fig. 4a-c. e, In vivo co-IP assay showing phosphate starvation induces Ada3 acetylation and promotes the association of Ada3-FLAG-containing SAGA and Ada3-Myc-containing SAGA. Diploid strains containing Ada3-FLAG/Ada3-Myc or Ada3-3KR-FLAG/Ada3-3KR-Myc were grown in SC media containing phosphate (SC + Pi) or phosphate free media (SC - Pi) for 3 hrs. Ada3-FLAG was immunoprecipitated with anti-FLAG beads. Both input and IP fractions were analyzed by western blots with the indicated antibodies. f, Regulation of Ada3 acetylation and SAGA dimerization by Rpd3 under normal or phosphate starvation medium. Diploid strains containing Spt7-TAP and Spt7-Myc were either grown in SC media containing phosphate (SC + Pi) or phosphate free media (SC - Pi) for 3 hrs. Spt7-TAP was immunoprecipitated with calmodulin beads. Both input and IP fractions were analyzed by western blots with the indicated antibodies. For Extended Data Fig. 4a-c, shown is the typical example of at least two biological independent experiments. For Extended Data Fig. 4e, f, shown is the typical example of three biological independent experiments.
Extended Data Fig. 5 The bromodomains of Gcn5 and Spt7 contribute to SAGA dimerization.
a, Amino acid sequences of the S. cerevisiae histone H3-K14ac and Ada3-K8ac, K14ac and K182ac. H3 lysine 14 and Ada3 lysine 8, 14 and 182 are highlighted in red. b, The molecular docking of Gcn5 bromodomain with Ada3 peptide acetylated at K182 (Ada3-EK182acR). c, The molecular docking of Spt7 bromodomain with Ada3 peptide acetylated at K8 (Ada3-GK8acL). d, The molecular docking of Spt7 bromodomain with Ada3 peptide acetylated at K14 (Ada3-EK14acL). e, Coomassie Blue staining of recombinantly purified Gcn5 bromodomain and Spt7 bromodomain. f, Isothermal titration calorimetry (ITC) assays were performed with purified recombinant Gcn5 bromodomain, Spt7 bromodomain and unacetylated Ada3 peptides. g, Silver staining of purified Wt SAGA, SAGA Gcn5 Bro-mut and Spt7 Bro-mut. h, Gcn5 bromodomain and Spt7 bromodomain are required for SAGA self-association as determined by in vivo Co-IP. i, Glycerol density gradient centrifugation analysis of Wt SAGA or SAGA Gcn5/Spt7 Bro-mut purified from cells grown in 2% sucrose-containing media. j, Quantification of fractions in Extended Data Fig. 5i. For Extended Data Fig. 5f, the quantitative data represent means ± SE (n=3). For Extended Data Fig. 5e, g, h, i, shown is the typical example of two biological independent experiments.
Extended Data Fig. 6 Ada3 acetylation-dependent SAGA dimerization promotes SAGA’s activity to acetylate nucleosomes.
a, Western blot analysis of histone modifications in WT, Ada3-K8R, Ada3-K14R, Ada3-K182R, Ada3-3KR, and ada3Δ mutants when grown in YP + 2% glucose (YP + Glu) or 2% sucrose (YP + Suc). b, Western blot analysis of histone modifications in WT and Gcn5/Spt7 Bro-mut when grown in YP + 2% glucose or 2% sucrose. c, Quantification of Western blot data in Fig. 6b-g. d, Comparison the HAT activity of SAGA purified from cells when grown in glucose (YP + Glu) and sucrose (YP + Suc) on nucleosome arrays. e, Silver staining of purified SAGA complexes used in Fig. 6b, e; Extended Data Fig. 6d. The acetylation level of Ada3 was determined by Western blots with a pan acetyl-lysine antibody. f, Comparison the HAT activity of SAGA monomers and SAGA dimers on mononucleosomes and dinucleosomes. g, Comparison the HAT activity of Wt SAGA and SAGA Ada3-3KR purified from glycerol gradient centrifugation on nucleosome arrays. h, Quantification of Fig. 6h. For Extended Data Fig. 6a-h, shown is the typical example of at least two biological independent experiments.
Extended Data Fig. 7 Ada3 acetylation-dependent SAGA dimerization is required for gene transcription under stress conditions.
a, Ada3 acetylation is required for PHO5 transcription when grown in phosphate starvation media. b, Bromodomains of Gcn5 and Spt7 are required for PHO5 transcription when grown in phosphate starvation media. c, GSEA analysis of cell cycle related genes in Ada3-3KR, Gcn5 Bro-mut and Spt7 Bro-mut. GSEA uses the expression information of all genes through clusterprofiler (R package). P value was calculated by permutation test based on the gene set. d, Flow cytometry analysis of cell cycle profiling of WT, Ada3-3KR and Gcn5/Spt7 Bro-mut when grown in glucose-containing medium. e, Volcano plots for differentially expressed genes by sucrose treatment from RNA-seq experiments. The edgeR is used to calculate P value. f, Ada3 acetylation is required for SUC2 transcription when grown in sucrose-containing media. For Extended Data Fig. 7a, b, f, data represent means ± SE; n=3 biological independent experiments; two-sided t-tests were used for statistical analysis.
Extended Data Fig. 8 Nucleosomes promote Ada3 acetylation by SAGA.
20 ng purified Wt SAGA was incubated with 0.5 mM acetyl-CoA at 30 °C for 0-30 min in the presence or absence of mononucleosomes. Shown is the typical example of two biological independent experiments.
Extended Data Fig. 9 Structural view of Gcn5 HAT catalytic domain with Ada3 acetylation sites.
The structure of Ada3 K8 (GK8L) (a), K14 (EK14L) (b), and K182 (EK182R) (c) peptides with Gcn5 HAT catalytic domain was generated from molecular dynamic simulation.
Extended Data Fig. 10 The interaction between SAGA and transcription activators and TBP is unaffected by Ada3 acetylation in vitro.
a GST pull-down assays with purified GST-Gal4 and purified WT SAGA and SAGA Ada3-3KR. GST only acts as a negative control. b GST pull-down assays with purified GST-Gcn4 and purified WT SAGA and SAGA Ada3-3KR. GST only acts as a negative control. c In vitro Co-IP with purified TBP and purified SAGA WT Ada3-FLAG and SAGA Ada3-3KR-FLAG. SAGA WT Ada3-FLAG and SAGA Ada3-3KR-FLAG were immunoprecipitated with anti-FLAG beads and TBP was detected with anti-TBP antibody. For Extended Data Fig. 10a-c, shown is the typical example of two biological independent experiments.
Supplementary information
Supplementary Tables 1–5
Supplementary Table 1: List of Kd values for Gcn5 bromodomain and Spt7 bromodomain to acetylated Ada3 peptides or unacetylated Ada3 peptides. Supplementary Table 2: RNA-seq analysis of DEGs in WT, Ada3-3KR, Gcn5 Bro-mut, Spt7 Bro-mut when treated with 2% glucose. Supplementary Table 3: RNA-seq analysis of DEGs in WT, Ada3-3KR, Gcn5 Bro-mut, Spt7 Bro-mut when treated with 2% sucrose. Supplementary Table 4: List of strains used in this study. Supplementary Table 5: List of oligonucleotides used in this study.
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Huang, J., Dai, W., Xiao, D. et al. Acetylation-dependent SAGA complex dimerization promotes nucleosome acetylation and gene transcription. Nat Struct Mol Biol 29, 261–273 (2022). https://doi.org/10.1038/s41594-022-00736-4
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DOI: https://doi.org/10.1038/s41594-022-00736-4
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