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Lysine acetylation of histone acetyltransferase adaptor protein ADA2 is a mechanism of metabolic control of chromatin modification in plants

Nature Plants volume 10, pages 439–452 (2024)Cite this article

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Abstract

Histone acetylation is a predominant active chromatin mark deposited by histone acetyltransferases (HATs) that transfer the acetyl group from acetyl coenzyme A (acetyl-CoA) to lysine ε-amino groups in histones. GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) is one of the best-characterized HATs and functions in association with several adaptor proteins such as ADA2 within multiprotein HAT complexes. ADA2–GCN5 interaction increases GCN5 binding to acetyl-CoA and stimulates its HAT activity. It remains unclear whether the HAT activity of GCN5 (which acetylates not only histones but also cellular proteins) is regulated by acetyl-CoA levels, which vary greatly in cells under different metabolic and nutrition conditions. Here we show that the ADA2 protein itself is acetylated by GCN5 in rice cells. Lysine acetylation exposes ADA2 to a specific E3 ubiquitin ligase and reduces its protein stability. In rice plants, ADA2 protein accumulation reversely parallels its lysine acetylation and acetyl-CoA levels, both of which are dynamically regulated under varying growth conditions. Stress-induced ADA2 accumulation could stimulate GCN5 HAT activity to compensate for the reduced acetyl-CoA levels for histone acetylation. These results indicate that ADA2 lysine acetylation that senses cellular acetyl-CoA variations is a mechanism to regulate HAT activity and histone acetylation homeostasis in plants under changing environments.

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Fig. 1: Rice ADA2 lysine acetylation by GCN5.
Fig. 2: ADA2 lysine acetylation promotes its ubiquitination by a specific E3 ligase.
Fig. 3: ADA2 protein, histone acetylation and acetyl-CoA levels in rice seedlings.
Fig. 4: ADA2 protein and lysine acetylation levels are influenced by mutations or stresses that reduce acetyl-CoA levels.
Fig. 5: ADA2 plays an important role in maintaining histone acetylation homeostasis in plant cells.
Fig. 6: Effect of acetyl-CoA concentration and ADA2 protein abundance on GCN5 HAT activity.

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Data availability

All data generated in this study are included in the main text. The structural models of ADA2 and E3-2 from the AlphaFold database and the modelling of the ADA2–E3-2 interaction are included in Supplementary Data 1. Source data are provided with this paper.

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Acknowledgements

We thank Q. Zhang and X. Li for technical assistance, H. Song for help with the confocal experiments and X. Zhang for advice on figure enhancement. Computation resources were provided by the high-throughput computing platform of the National Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University and supported by H. Liu. The work was supported by National Natural Science Foundation of China (grant no. 32070563, D.-X.Z.), the Fundamental Research Funds for the Central Universities (grant no. 2662015PY228, D.-X.Z.) and the French Agence Nationale de Recherche project (grant no. REPHARE ANR-19-CE12-0027, D.-X.Z.).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Yue Yu, Feng Zhao, Yaping Yue, Yu Zhao & Dao-Xiu Zhou

  2. Institute of Plant Sciences Paris-Saclay (IPS2), CNRS, INRAE, University Paris-Saclay, Orsay, France

    Dao-Xiu Zhou

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Contributions

Y. Yu did the experimental work and data analysis. F.Z. participated in the construction of transgenic plants. Y. Yue and Y.Z. contributed to the experimentation. D.-X.Z. designed and supervised the work and wrote the paper with input from Y. Yu.

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Correspondence to Dao-Xiu Zhou.

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Nature Plants thanks Chenlong Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1. Representative MS/MS spectra of 7 acetylpeptides of ADA2.

SPSHIAGGANK(ac)K, ASNVGQFK(ac)DGANVAK, DGANVAK(ac)VEDGHVDR,SIGVK(ac)KPR, YSADEGPSLTELSGYNSK(ac)R, ESGQLLSNTK(ac)VVHK, IESDGNLDQK(ac)K, with an acetylation site at K of ADA2 respectively. The x axis represents the mass charge ratio (m/z), and the y axis represents the relative abundance of ions.

Extended Data Fig. 2 Lysine acetylation effects on ADA2 function.

(a) Protein interaction between ADA2 full-length, truncated fragments, or the K263R point mutation version and GCN5 in yeast two hybrid assays. (b) Effects of the point mutations (K263R and K263Q) for ADA2 nucleus localization in rice protoplasts. At least two repetitions were performed. (c) Tests of ADA2 function to promote the acetyltransferase activity of GCN5. In vitro HAT assay was performed using recombinant GCN5-His as acetyltransferases and recombinant H3.3-His as substrates with or without ADA2-His recombinant protein in 50 µM acetyl-CoA. GCN5E233AD274A-His is GCN5 catalytically dead mutant, GCN5Y485A-His is a bromodomain mutation that disrupts acetyl-lysine binding. (d) Tests of effect of the ADA2 7KR point mutations on acetyltransferase activity of GCN5. In vitro HAT assay was performed using recombinant GST-GCN5 as acetyltransferases and recombinant H3.3-His as substrates with or without ADA2-His or ADA27KR-His recombinant protein in 50 µM Acetyl-CoA. The assays in c and d were analyzed by immunoblotting with anti-H3, anti-pan-H3 acetylation, anti-H3K9 acetylation, anti-H3K14 acetylation, anti-acetyl-lysine and anti-ADA2 antibody. Data in c and d are the two repetitions (except the controls used in c and d are different).

Source data

Extended Data Fig. 3 Detrimental effect of ADA2 acetylation on its stability.

The two other replicates of Fig. 2a.

Source data

Extended Data Fig. 4 Identification of a specific E3 ligase that targets acetylated ADA2 in rice.

(a) Network view of predicted E3-substrate interactions in UbiBrowser web services. In order to found the candidate E3 ligase we used ADA2 homologs gene TADA2b (Homo sapiens) as queried substrate to predicted potential E3 ligase. In network view, the central node is the queried substrate, and the surrounding nodes are the predicted E3 ligases. The width of the edge reflects the confidence of the interaction. (b) Predicted high confidence (green background) and middle confidence (yellow background) interacting E3 proteins in Homo sapiens, and their homologs in rice. (c) Co-localization of E3-2 (fused to RFP) with wild type and mutant ADA2 proteins (fused to GFP) in transiently transfected rice protoplasts. (d) Visualization of the ZDOCK predicted ADA2 and E3-2 interacting residues. The distances between E3-2 and ADA2 or ADA2 K263Q mutant residues were measured using pymol software. (e) Production of rice E3-2 CRISPR/Cas9 mutants. sgRNA position in the gene and decoded sequence mutations of the E3-2 gene are shown. (f) Phenotypes of e3-2-1 and e3-2-2 plants in comparison with a control line (with null mutation) at seedling stage. (g) ADA2 protein levels in e3-2 mutants relative to the control line. Nuclear proteins were tested by immunoblotting with anti-ADA2 and anti-H3 antibodies. Band intensities were quantified using ImageJ software, relative to the control line, and indicated below the blots.

Source data

Extended Data Fig. 5 ADA2 protein is accumulated during the dark period.

(a) Nuclear proteins were extracted from rice seedlings (14 d after germination), grown on a 12-h diurnal light/dark cycle sampled at 6-h intervals from ZT12 to ZT6 for 2 days or with continuous darkness as indicated, followed by immunoblotting with anti-ADA2 and anti-H3 antibodies. (b) Endogenous ADA2 was immunoprecipitated from the nuclear proteins prepared as in (a) with anti-ADA2 antibody, followed by immunoblotting with anti-acetyl-lysine (anti-Lys-Ac) antibody to detect ADA2 acetylation. (c) The other replicate of Fig. 3A. (d) ADA2 transcript levels in rice seedlings indicated in (a). Transcript levels (n = 3, mean values from 3 measurements are presented) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene.

Source data

Extended Data Fig. 6 Time course of ADA2 protein accumulation under stress.

(a) ADA2 protein and transcript levels under PEG stress. Upper panels: Immunoblotting analysis of ADA2 protein levels in rice seedlings treated with or without PEG during the day time, harvested at 3 h intervals. Anti-H3 was used as loading controls. Lower part: relative ADA2 transcript levels during PEG treatment. ADA2 transcript levels in rice seedlings (n = 3, mean values from 3 measurements are presented) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene. (b) ADA2 protein and transcript levels in seedlings treated with 20% PEG6000 for 24 h with or without ActD (40 µM) treatment. Nuclear proteins were analyzed by immunoblotting with anti-ADA2 and anti-H3 antibodies. Band intensities were quantified using ImageJ software, relative to CK, and indicated below the blots. Two replicates (rep 1 &2) are shown. ADA2 transcript levels in rice seedlings (bars indicate the mean ± SE of three replicates) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene. (c) ADA2 protein levels in rice seedling (10 days after germination) under submergence. Rice plant samples were harvested at ZT6 every 24 hours during 7 days, for acetyl-CoA and nuclear protein extraction, followed by immunoblotting with anti-ADA2 and anti-H3 acetylation antibodies. ADA2 transcript levels in rice seedlings (bars indicate the mean ± SE of three replicates) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene.

Source data

Extended Data Fig. 7 ADA2 is required for PEG stress tolerance and expression and histone acetylation of stress responsive genes.

(a) Drought sensitive phenotypes of wild-type and ada2 mutant plants. Rice seedlings of the same size/length were dehydrated for 4 days and then rehydrated for 2 days. The average percentage of survival were calculated from two independent experiments with at least 13 plants of each genotype in each replicate. (b) WT and ada2-1 mutant seedlings were treated with or without 20% PEG6000 for 24 h. The H3K14 acetylation levels of the stress mark genes were measured by Cut&tag-qPCR, enrichment values represent the relative fold change of H3K14 acetylation antibody to IgG antibody. Transcript levels of the stress mark genes in rice seedlings were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using actin as a reference gene. SUS4 was used as negative control. For all data, bars indicate the mean ± SE of three replicates (Unpaired and two-tailed t test, the significance level was p < 0.05). (c) The WT seedlings were treated with or without 20% PEG6000 in the absence of MG132 for 24 h. H3K14 acetylation and transcript levels of the stress-responsive genes were measured as in B, and the p-value between the two groups is shown above the column, bars indicate the mean ± SE of three replicates (Unpaired and two-tailed t test, the significance level was p < 0.05).

Supplementary information

Supplementary Information

The primer sequences used in this study.

Reporting Summary

Supplementary Data 1

The structural models of ADA2 and E3-2 from the AlphaFold database and the modelling of the ADA2–E3-2 interaction.

Source data

Source Data Fig. 1

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Source Data Figs. 2–5 and Extended Data Figs. 5–7

Statistical source data for Figs. 2–5 and Extended Data Figs. 5–7.

Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 3

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Yu, Y., Zhao, F., Yue, Y. et al. Lysine acetylation of histone acetyltransferase adaptor protein ADA2 is a mechanism of metabolic control of chromatin modification in plants. Nat. Plants 10, 439–452 (2024). https://doi.org/10.1038/s41477-024-01623-0

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