Abstract

Human p300 is a transcriptional co-activator and a major acetyltransferase that acetylates histones and other proteins facilitating gene transcription. The activity of p300 relies on the fine-tuned interactome that involves a dozen p300 domains and hundreds of binding partners and links p300 to a wide range of vital signaling events. Here, we report a novel function of the ZZ-type zinc finger (ZZ) of p300 as a reader of histone H3. We show that the ZZ domain and acetyllysine-recognizing bromodomain of p300 play critical roles in modulating p300 enzymatic activity and its association with chromatin. The acetyllysine binding function of bromodomain is essential for acetylation of histones H3 and H4, whereas interaction of the ZZ domain with H3 promotes selective acetylation of the histone H3K27 and H3K18 sites.

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Acknowledgements

We thank J. Nix at beam line 4.2.2 of the ALS in Berkeley for help with X-ray crystallographic data collection. This work was supported by grants from NIH GM106416, GM125195, and GM100907 to T.G.K., CA204020 to X.S., and HG007538 and CA193466 to W.L., and from the Cancer Prevention and Research Institute of Texas (RP160237 and RP160739), the Welch Foundation (G1719), and the Leukemia & Lymphoma Society Career Development Program Scholarship to X.S.

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Author notes

  1. These authors contributed equally: Y. Zhang, Y. Xue.

Affiliations

  1. Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA

    • Yi Zhang
    • , JaeWoo Ahn
    • , Muzaffar Ali
    • , Brianna J. Klein
    •  & Tatiana G. Kutateladze
  2. Department of Epigenetics and Molecular Carcinogenesis, Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Yongming Xue
    • , Wenyi Mi
    • , Xiaolu Wang
    • , Hong Wen
    •  & Xiaobing Shi
  3. Genetics and Epigenetics Graduate Program, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA

    • Yongming Xue
    •  & Xiaobing Shi
  4. Dan L. Duncan Cancer Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

    • Jiejun Shi
    •  & Wei Li
  5. Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, USA

    • Wenyi Mi
    • , Xiaolu Wang
    • , Hong Wen
    •  & Xiaobing Shi

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Contributions

Y.Z., Y.X., J.S., J.W.A., W.M., M.A., X.W., B.J.K., and H.W. performed experiments and together with W.L., X.S., and T.G.K. analyzed the data. Y.Z., X.S., and T.G.K. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

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Correspondence to Xiaobing Shi or Tatiana G. Kutateladze.

Integrated supplementary information

  1. Supplementary Figure 1 The ZZ domain of p300 binds the histone H3 tail.

    a, Superimposed 1H,15N HSQC spectra of p300 ZZ collected upon titration with H3K4ac, H3K4me1 and H3K4me3 peptides (residues 1–12 of histone H3). Spectra are color-coded according to the protein:peptide molar ratio. b, Superimposed 1H,15N HSQC spectra of p300 ZZ (1668–1713) collected upon titration of histone H3 (1–12) peptide. Spectra are color-coded according to the protein:peptide molar ratio as in a.

  2. Supplementary Figure 2 Binding affinities of the indicated domains of p300 to histone H3 tail peptides.

    a, Binding curves used to determine the Kd values by fluorescence. b, Fitting the fluorescence data for the interaction of WT HAT-ZZ with H31–31 using models with one and two binding sites.

  3. Supplementary Figure 3 NMR spectra of the ZZ constructs.

    Superimposed 1H,15N HSQC spectra of the linked H3-ZZ construct (blue) and either the isolated ZZ domain in the presence of a tenfold excess of H31–12 peptide (black) or the apo state of ZZ (red).

  4. Supplementary Figure 4 Structural comparison of the ZZ domain of p300 with other readers of the H3 tail.

    a, Crystal structure of the linked H3-ZZ construct. b, The ZZ domain structure and the H3-binding mode are distinctly different from the structures and H3-binding modes of other known H3-specific readers. An overlay of the structures of p300-ZZ in complex with H3 and the PHD finger of BHC80 in complex with H3 (PDB 2PUY; Nature 448, 718–722, 2007) reveals a unique, non-superimposable fold of each of these readers. c, Structural comparison of readers recognizing the N-terminal sequence of histone H3. Shown are complexes of the PHD finger of BHC80 (PDB 2PUY; Nature 448, 718–722, 2007), the WD40 domain of Nurf55 (PDB 2YBA; Mol. Cell 42, 330–341, 2011), and the ADD domain of DNMT3L (PDB 2PVC; Nature 448, 714–717, 2007) with unmodified histone H3 peptides. Unmodified K4 of histone H3 (H3K4) is restrained through multiple-hydrogen bonding and electrostatic contacts in the complexes. Binding of these readers is abrogated by methylation of H3K4. In contrast, the p300 ZZ domain is insensitive to PTMs on H3K4. Other histone H3 residues, including A1, R2 and Q5, are uniquely constrained in the p300-ZZ:H3 complex (Fig. 2a, b).

  5. Supplementary Figure 5 Recruitment of p300 to chromatin requires the histone-binding functions of both ZZ and BD.

    a, Calf thymus histone pulldown assays of the p300 ZZ domain. RBP2 PHD1 and GST were used as a positive and negative control, respectively. b, Calf thymus histone pulldown assays of the wild-type p300 ZZ domain and the indicated mutants. c,d, Salt fractionation of H1299 cells stably expressing wild-type or point- and deletion-mutated FLAG-p300BRPHZT. The workflow for the salt fractionation assay is shown. e, Western blot analysis showing that chromatin in ChIP–Western and salt fractionation assays contains histone acetyl-lysine marks.

  6. Supplementary Figure 6 The relationship of the adjacent to the HAT domain modules.

    a, Superimposed 1H,15N HSQC spectra of p300 BD-RING-PHD collected upon titration with the indicated histone peptides. Spectra are color-coded according to the protein:peptide molar ratio. b, Superimposed 1H,15N HSQC spectra of p300 BD-RING-PHD collected upon titration with HAT or HAT-ZZ. Spectra are color-coded according to the protein:ligand molar ratio. c, Superimposed 1H,15N HSQC spectra of p300-ZZ (red) and HAT-ZZ (black).

  7. Supplementary Figure 7 ZZ modulates the HAT activity of p300.

    a, Western blot analysis showing equal amounts of p300BRPHZ fragments used in the HAT assays. b, Western blot analysis of histone acetylation levels in whole-cell extract of 293T cells transiently expressing full-length p300 or the mutants. c, Representative genome browser views of the FLAG (brown), H3K18ac (blue) and H3K27ac (purple) ChIP-seq signals on the indicated genes. The black lines indicate the sites for ChIP–qPCR. d, A model of the p300 BD-RING-PHD-HAT-ZZ region in complex with H3 (1–31). The electrostatic surface potential of p300 HAT is colored blue (positive charge) and red (negative charge) with the histone H3 peptide shown in black. The H3-binding site of ZZ and the active site of HAT are indicated by cyan circles. In this model, the distance between Ala1 of H3 bound by ZZ and the active site of HAT is ~38 Å. An initial structure of p300 BD-RING-PHD-HAT (PDB 4BHW) was used in modeling.

  8. Supplementary Figure 8 Comparison of binding activities of the ZZ domains of p300 and CBP.

    a, Superimposed 1H,15N HSQC spectra of the same length (51-residue) constructs of p300-ZZ (top) and CBP-ZZ (bottom) collected upon titration with H3 (1–12) peptide. b, Superimposed 1H,15N HSQC spectra of the same length (46-residue) constructs of p300-ZZ (top) and CBP-ZZ (bottom) collected upon titration with H3 (1–12) peptide. c, Overlays of 1H,15N HSQC spectra of p300-ZZ (left) and CBP-ZZ (right) recorded upon titration with SUMO1. No CSPs were observed in either experiment, indicating that the p300/CBP ZZ domains do not interact with SUMO1. d, Structural overlay of p300-ZZ without H3 and apo CBP-ZZ (PDB 1TOT) suggests that the Ala1-binding site in CBP is partially occluded.

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https://doi.org/10.1038/s41594-018-0114-9