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Structure of the p300 catalytic core and implications for chromatin targeting and HAT regulation

Nature Structural & Molecular Biology volume 20, pages 1040–1046 (2013)Cite this article

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Abstract

CBP and p300 are histone acetyltransferases (HATs) that associate with and acetylate transcriptional regulators and chromatin. Mutations in their catalytic 'cores' are linked to genetic disorders, including cancer. Here we present the 2.8-Å crystal structure of the catalytic core of human p300 containing its bromodomain, CH2 region and HAT domain. The structure reveals that the CH2 region contains a discontinuous PHD domain interrupted by a RING domain. The bromodomain, PHD, RING and HAT domains adopt an assembled configuration with the RING domain positioned over the HAT substrate-binding pocket. Disease mutations that disrupt RING attachment led to upregulation of HAT activity, thus revealing an inhibitory role for this domain. The structure provides a starting point for understanding how chromatin-substrate targeting and HAT regulation are coupled and why mutations in the p300 core lead to dysregulation.

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Figure 1: Structure of the p300 core.
Figure 2: Domains of the p300 core.
Figure 3: Structural details and analysis of interaction interfaces.
Figure 4: Effects of mutations on HAT activity and chromatin binding of p300.
Figure 5: Model for p300 autoregulation and substrate acetylation.

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Acknowledgements

We thank P.A. Cole (Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) for supply of Lys-CoA and S.C. Harrison and C. Petosa as well as members of the Panne laboratory for comments on the manuscript. We thank the staff of the European Synchrotron Radiation Facility and European Molecular Biology Laboratory–Grenoble for assistance and support in using beamlines ID14-4, 23-1 and 23-2. This work used the platforms of the Grenoble Instruct Centre (Integrated Structural Biology, Grenoble; UMS 3518 CNRS-CEA-UJF-EMBL) with support from the French Infrastructure for Integrated Structural Biology (ANR-10-INSB-05-02) and Grenoble Alliance for Integrated Structural Cell Biology (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology. This work was supported by the Agence Nationale de la Recherche Blanc Grant 'EPISTRUCT' (D.P.).

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  1. Manuela Delvecchio

    Present address: Present address: Diasorin Società per Azioni, Saluggia, Italy.,

Authors and Affiliations

  1. European Molecular Biology Laboratory, Grenoble, France

    Manuela Delvecchio, Jonathan Gaucher, Carmen Aguilar-Gurrieri, Esther Ortega & Daniel Panne

  2. Unit for Virus Host-Cell Interactions, University of Grenoble Alpes, European Molecular Biology Laboratory–Centre National de la Recherche Scientifique, Grenoble, France

    Manuela Delvecchio, Jonathan Gaucher, Carmen Aguilar-Gurrieri, Esther Ortega & Daniel Panne

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Contributions

M.D., J.G., C.A.-G. and E.O. designed and performed the experiments. D.P. advised and assisted in all aspects of the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Daniel Panne.

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Integrated supplementary information

Supplementary Figure 1 Biochemical characterization.

(a) Mass spectrometry analysis using electrospray ionization (ESI) of the p300 'core' before, and after (b) deacetylation by SIRT2. Inset shows a Western blot of p300 deacetylation by SIRT2. Levels of acetylation were detected by western blot using an anti–acetyllysine antibody. Incubation times in hours (h) are indicated above the gel. After overnight (o/n) incubation, SIRT2 was able to completely deacetylate the p300 'core'. (c) HAT activity was measured with a variant in which the AIL was replaced with a 5 residue linker (Δ1520–1581). Substrate was a peptide derived from histone H3 (1–20) ARTKQTQRKSTGGKAPRKQL.

Supplementary Figure 2 Structural comparisons of PHD and RING domains.

(a) Structure of the BPTF PHD domain bound to H3K4me3. (b) Superimposed structures of p300 PHD (red), Pygo (2VPE, green), BPTF (3qvz, orange), ING2 (2G6Q, blue). The PHD domain is most similar to the PHD domain of BPTF. (c) Structure–based sequence alignment of the PHD domain. Zinc ligand positions are shown as red boxes. Residues that make up the aromatic cage are shown as a blue box and labeled I–IV. Residues that make specific contacts with the H3K4me2/3 peptide are shown in green. (d) The RING domain is similar to other RING structures. Superimposed structures of p300 RING (green) and Cbl RING domain (red). The p300 RING domain is missing the second Zinc binding site. The sequence insertion in loop L1 is shown. (e) Structure–based sequence alignment of the RING domain. Zinc ligand positions as above. Residues that make specific contacts with E2 enzymes are shown in blue. 3MMK: Residues 56–290 are omitted from the alignment.

Supplementary Figure 3 BRP-HAT interaction and structural details and interaction interfaces.

(a), The BRP module binds to the HAT domain. Pull–down experiments with GST–BRP against the HAT domain. Input (I), Wash (W) and Bound (B). (b) Isothermal titration calorimetry–based binding curves with wild–type BRP or a Bromodomain mutant BRP N1132. (c) Stereo view of the RING–HAT interface. (d) Crystal packing in the vicinity of the RING domain. The HAT domain (HAT*) of a crystallographically related molecule packs against the RING domain. (e) There are two p300 molecules in the asymmetric crystallographic unit; they overlay well on each other (rmsd 0.9 Å for 564 Ca residues). Coloring as in Figure 1.

Supplementary Figure 4 Multiple sequence alignment of p300 and CBP orthologs.

Human (hp300), mouse (mp300), bovine (bp300) and dog (cp300). Also included are the CBP homologs human (hCBP), mouse (mCBP), rat (rCBP), Drosophila melanogaster (dCBP) and Caenorhabditis elegans (cCBP). Identical residues are shown with a red background and similar residues with red letters. Secondary structure elements of p300 are shown above the amino acid sequences. The Bromodomain (BROMO) is highlighted in yellow, the RING domain in green, the PHD domain in red and the HAT domain in blue. Intramolecular contacts between domains: Residues in the BROMO–PHD interface (•). Residues in the RING–HAT interface (Δ). Residues in the PHD–HAT interface (*). The sequence alignment was performed using ClustalW1 and the figure generated using ESPript2.

Supplementary Figure 5 Functional analysis of the RING domain and in vivo assays.

(a) Superposition of E2–ubiquitin onto the RING domain of p300. The ternary complex of RNF4:UbcH5–ubiquitin (Protein Data Bank entry 4AP4) was superimposed onto the RING domain of p300. Residues of the p300 RING and HAT domains that would sterically clash with UbcH5 or Ubiquitin if the interaction were to follow the mode of the RNF4:UbcH5–ubiquitin complex are colored in red. (b) E3 ligase activity assays. Polyubiquitination assays were done using the indicated p300 constructs and the E2 conjugating enzymes UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10 and UbcH13 complex. The result obtained with UbcH5a is shown here. Reactions were incubated for 1 h at 37 °C followed by SDS–PAGE and immunoblotting. (c-f) H1299 cells were transfected with the indicated expression vectors (c) GFP, (d) GFP–BRP, (e) GFP–BRP N1132A, (f) GFP–Bd. Cells were treated with or without the HDAC inhibitor Trichostatin A (TSA). Ectopically expressed proteins were visualized by GFP and co-detected with an anti-tetraacetyl-Histone H4 antibody. Immunofluorescence was performed with anti-HA or anti-H3K56ac antibody. Bar: 10 μm.

Supplementary Figure 6 Uncropped images for the blots shown in Figure 4a,b.

(a) Anti–p300 K1499 acetyl immunoblot of lysates of H1299 cells transfected with the indicated mutants. (b) Loading control of hemagglutinin (HA)-tagged p300, determined by immunoblotting with anti-HA antibody. (c) Anti–p53 K373 K382 acetyl (p53 KAc) immunoblot of lysates of H1299 cells, transfected with the indicated p300 mutants and p53. (d) Loading control of Flag-tagged p53 and (e) HA-p300, determined by immunoblotting with anti-Flag or anti-HA antibody, respectively.

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Delvecchio, M., Gaucher, J., Aguilar-Gurrieri, C. et al. Structure of the p300 catalytic core and implications for chromatin targeting and HAT regulation. Nat Struct Mol Biol 20, 1040–1046 (2013). https://doi.org/10.1038/nsmb.2642

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