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Acetylation-regulated interaction between p53 and SET reveals a widespread regulatory mode

Abstract

Although lysine acetylation is now recognized as a general protein modification for both histones and non-histone proteins1,2,3, the mechanisms of acetylation-mediated actions are not completely understood. Acetylation of the C-terminal domain (CTD) of p53 (also known as TP53) was an early example of non-histone protein acetylation4 and its precise role remains unclear. Lysine acetylation often creates binding sites for bromodomain-containing ‘reader’ proteins5,6. Here we use a proteomic screen to identify the oncoprotein SET as a major cellular factor whose binding with p53 is dependent on CTD acetylation status. SET profoundly inhibits p53 transcriptional activity in unstressed cells, but SET-mediated repression is abolished by stress-induced acetylation of p53 CTD. Moreover, loss of the interaction with SET activates p53, resulting in tumour regression in mouse xenograft models. Notably, the acidic domain of SET acts as a ‘reader’ for the unacetylated CTD of p53 and this mechanism of acetylation-dependent regulation is widespread in nature. For example, acetylation of p53 also modulates its interactions with similar acidic domains found in other p53 regulators including VPRBP (also known as DCAF1), DAXX and PELP1 (refs. 7, 8, 9), and computational analysis of the proteome has identified numerous proteins with the potential to serve as acidic domain readers and lysine-rich ligands. Unlike bromodomain readers, which preferentially bind the acetylated forms of their cognate ligands, the acidic domain readers specifically recognize the unacetylated forms of their ligands. Finally, the acetylation-dependent regulation of p53 was further validated in vivo by using a knock-in mouse model expressing an acetylation-mimicking form of p53. These results reveal that acidic-domain-containing factors act as a class of acetylation-dependent regulators by targeting p53 and, potentially, other proteins.

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Figure 1: Identification of SET as a specific co-repressor of C-terminal unacetylated p53.
Figure 2: SET negatively regulates p53 transactivity by inhibiting p300/CBP-mediated H3K18 and H3K27 acetylation on the p53 target promoter.
Figure 3: Acidic-domain-containing proteins represent a new class of ‘reader’ for their unacetylated ligands.
Figure 4: The physiological significance of acetylation-dependent dissociation of p53 from its acidic-domain-containing ‘readers’.

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Primary accessions

Gene Expression Omnibus

Data deposits

RNA-seq data is available through NCBI Gene Expression Omnibus (GEO) database with the accession number GSE83635.

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Acknowledgements

We thank F. Giancotti, X. Yang, R. K. Vadlamudi and W. An for providing reagents for this work. We also thank R. Baer for discussion and suggestions. This work was supported by the National Cancer Institute of the National Institutes of Health under Award 5R01CA193890, 5RO1CA190477, 5RO1CA085533 and 2P01CA080058 to W.G. and GM030518 and CA121852 to B.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

Authors

Contributions

The experiments were conceived and designed by D.W., N.K., G.L. and W.G. The experiments were performed mainly by D.W. and N.K. Bioinformatic analysis was performed by G.L. Mass spectrometry analysis was performed by W.L. The xenograft assay was performed by D.W. and L.J. Data were analysed and interpreted by D.W., N.K., G.L., W.-G.Z., J.Q., B.H. and W.G. The manuscript was written by D.W., N.K., G.L. and W.G.

Corresponding author

Correspondence to Wei Gu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Further analysis of p53–SET interaction.

a, A list of SET peptides identified by mass spectrometry. b, In vitro binding assay of methylated p53 CTD and purified SET. ce, In vitro binding assay between SET and the purified ubiquitinated, sumoylated or neddylated forms of p53. f, g, Western blot analysis of p53 and SET domains for their interaction. In vitro binding assay was performed by incubating immobilized GST, GST–p53 or GST–SET with each purified SET or p53 protein, as indicated. h, Western blot analysis of the interaction between p53 and SET in cells. H1299 cells were co-transfected with indicated constructs and the nuclear extract was analysed by co-IP assay. ik, ChIP analysis of p53 or SET recruitment onto the PUMA (i), TIGAR (j) or GLS2 (k) promoter. HCT116 cells were treated with or without 1 μM doxorubicin for 24 h and then the cellular extracts were analysed by ChIP assay with indicated antibodies. Asterisks indicate the specific bands of indicated proteins. Error bars indicate mean ± s.d., n = 3 for technical replicates. Data are shown as representative of three experiments. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 2 RNA-seq analysis to identify genes regulated by p53–SET interplay.

a, Western blot analysis of the expression of p53 in U2OS-derived CRISPR control cells or CRISPR p53-KO cells. b, Heat map of genes regulated by the p53–SET interplay. U2OS (CRISPR Ctr or CRISPR p53-KO) cells were transfected with control siRNA or SET-specific siRNA for 4 days and the total RNA was prepared for RNA-seq analysis with two or three biological replicates, as indicated. Known p53 target genes which were also repressed by SET in a p53-dependent manner were selected and presented as a heat map. The relative SET expression is shown in the last row of the heat map. c, qPCR validation of the genes regulated by the p53–SET interplay. Error bars indicate mean ± s.d., n = 3 for technical replicates. Data are shown as representative of three experiments. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 3 SET-mediated effects on cell proliferation and tumour growth.

a, b, Representative image (a) or quantitative analysis (b) of the SET knockdown-mediated effect on cell growth of U2OS-derived CRISPR control cells or CRISPR p53-KO cells. c, Western blot analysis of the expression of p53 in HCT116-derived CRISPR control cells or CRISPR p53-KO cells. d, Xenograft analysis of the SET-mediated effect on tumour growth by HCT116-derived CRISPR control cells or CRISPR p53-KO cells. e, Western blot analysis of p53 expression in control or derived HCT116 cell lines, as indicated. Error bars indicate mean ± s.d., n = 3 in b or n = 5 in d for biological replicates. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 4 SET regulates histone modifications on p53 target promoter.

a, Western blot analysis of the SET knockdown-mediated effect on the p53 C-terminal acetylation in HCT116 cells. Doxorubicin (Dox)-treated cells were also analysed in parallel as a positive control. b, Western blot analysis of the SET-mediated effect on the CBP-induced p53 C-terminal acetylation in H1299 cells. c, e, ChIP analysis of promoter-recruitment of p53 (c) or p300/CBP (e) upon SET depletion in HCT116 cells. d, ChIP analysis of the SET-knockdown-mediated effect on histone modifications in the PUMA promoter in HCT116 cells. f, ChIP analysis of the SET-mediated effect on p53-dependent H3K18 and H3K27 acetylation in the PUMA promoter. Error bars indicate mean ± s.d., n = 3 for technical replicates. Data are shown as representative of three experiments. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 5 Acetylation regulates the interaction between acidic-domain-containing proteins and their acetylatable ligands.

a, A summary table of characteristic features of the acidic-domain-containing proteins SET, VPRBP, DAXX and PELP1. The acidic amino acids are underlined. b, In vitro binding assay of p53 CTD and purified full-length VPRBP, DAXX or PELP1. ce, Western blot analysis of the interaction between p53 and VPRBP (c), DAXX (d) or PELP1 (e) in the nuclear fraction of H1299 cells. fh, In vitro binding assay between purified SET and KRD of H3 (f), KU70 (g) or FOXO1 (h). i, In vitro binding assay of the H3 KRD and purified VPRBP, DAXX or PELP1. j, In vitro binding assay of the H3 KRD and BRD4 or BRD7 (nuclear extract). Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 6 p53KQ mutant mimics acetylated p53.

a, Schematic diagram of human unacetylated p53 and the acetylation-deficient and acetylation-mimicking mutants of p53. b, In vitro binding assay of SET and different types of p53, as indicated. ce, Western blot analysis of the interaction between acidic-domain-containing proteins (c, VPRBP; d, DAXX; e, PELP1) and different types of p53 in cells. H1299 cells were co-transfected with indicated constructs, and the nuclear extract was analysed by Co-IP assay. Asterisks indicate the purified proteins. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 7 Generation of p53KQ/KQ mice.

a, Schematic diagram of the gene targeting strategy to replace the p53 C-terminal 7 lysines with 7 glutamines in mouse p53. b, Southern blot screening of ES cells to identify p53+/KQ clones. c, PCR genotyping analysis of wild-type (110 bp), p53+/KQ heterozygous (110 bp and 150 bp), and p53KQ/KQ homozygous mice (150 bp only). d, Sequencing analysis of the transcripts prepared from the p53+/KQ heterozygous mouse spleen. e, A summary table of observed numbers of mice from p53+/KQ heterozygous intercrosses. f, Positive control for p53 staining in the IHC assay. The spleen tissue sections of p53+/+ mice treated with or without 6 Gy γ-radiation was stained with p53 (CM-5) antibody. g, h, Representative image (g) or quantitative analysis (h) of SET-knockdown-mediated cell growth of p53+/+ or p53KQ/KQ MEFs (P2). Error bars indicate mean ± s.d., n = 3 for biological replicates. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Figure 8 Characterization of Set conditional knockout mice.

a, Schematic diagram of the strategy to generate Set conditional knockout mice. b, Validation of Set knockout in embryos (E8.5) by genotyping and western blot analysis. c, A summary table of observed numbers of embryos or pups from Set+/− intercrosses. d, Representative pictures of Set+/+ and Set−/− embryos (E10.5). e, qPCR analysis of the expression of p53 target genes in Set+/+ and Set−/− embryos (E10.5). Error bars indicate mean ± s.d., n = 3 for technical replicates. Data are shown as representative of three experiments. Uncropped blots can be found in Supplementary Fig. 1.

Extended Data Table 1 A list of human proteins containing acidic domains with a minimum percentage of acidic residues of 76% within a 36-residue window
Extended Data Table 2 A list of human proteins containing KRDs with at least five lysines where three or more lysines are annotated as acetylation sites in the SSPKA database

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Wang, D., Kon, N., Lasso, G. et al. Acetylation-regulated interaction between p53 and SET reveals a widespread regulatory mode. Nature 538, 118–122 (2016). https://doi.org/10.1038/nature19759

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