Fig 1: Signal changes of the H3 N-tail due to H4 acetylation in the NCP and nucleosome(A) Expanded spectra of 11 residues of the H3 N-tail, for which minor peaks appeared after H4 acetylation in the nucleosome. Shown are signals of the NCP (blue), H4ac-NCP (green), nucleosome (red), and H4ac-nucleosome (pink). Centers of the major and minor signals are marked with a circle and triangle, respectively.(B) Backbone {1H}-15N heteronuclear NOE values of the H3 N-tail in the NCP (blue) and H4ac-NCP (green) at 25 mM NaCl. Symbols indicate singlet signal (filled circle), high-field side of doublet signal (filled triangle), and low-field side of doublet signal (filled square). Error bars were calculated based on the signal-to-noise ratio.(C) Comparison of K14 acetylation rate by time-resolved NMR changes of A15 in the NCP (blue circle), H4ac-NCP (green circle), nucleosome (red circle), and H4ac-nucleosome (pink circle). Gcn5 enzyme was first added at 0.1 μM and increased to 1 μM after 20 h. Signal intensities were normalized by the initial signal recorded before the addition of Gcn5. Blue, green, red, and pink lines represent the data fit to the exponential equation for the NCP, H4ac-NCP, nucleosome, and H4ac-nucleosome, respectively.(D) DNA interaction model of the H3 N-tail (blue) and H4-acetylated N-tail (green) in the H4ac-NCP. Regardless of whether the H4 N-tail is acetylated or not, the H3 N-tail fluctuates dynamically between contact and non-contact states with the core DNA (PDB: 5av6).(E) DNA interaction model of the H3 N-tail (blue) and H4-acetylated N-tail (green) in the H4ac-nucleosome. The H3 N-tail in the H4ac-nucleosome makes dynamic contacts with DNA at a location that was previously occupied by the H4 N-tail (PDB: 7K61).See also Figure S3.
Fig 2: Comparison of H3 K14 acetylation between the NCP and the nucleosome(A) Signal changes of K14 and acetylated K14 of the H3 N-tail in the NCP (blue) and nucleosome (red) after the addition of Gcn5.(B) Comparison of K14 acetylation rate in the NCP (blue circle) and nucleosome (red circle) by time-resolved NMR spectroscopy. Signal intensities were normalized by the initial signal measured before the addition of Gcn5. Blue and red lines represent the data fit to the exponential equation for the NCP and nucleosome, respectively.(C) Superposition of 1H–15N HSQC spectra of the H3 N-tail in the NCP (blue) and nucleosome (red), and the K14-acetylated H3 N-tail in the NCP (green) and nucleosome (pink) in 25 mM MES (6.0), 25 mM NaCl, and 2 mM DTT.(D) Chemical shift differences of H3 N-tail residues between the NCP and nucleosome (blue), and K14-acetylated H3 N-tail residues between the NCP and nucleosome (green) at 25 mM NaCl. Symbols indicate singlet signal (filled circle), high-field side of doublet signal (filled triangle), and low-field side of doublet signal (filled square).
Fig 3: Carnosol is a specific p300 inhibitor in vitro. (A) Carnosol inhibit histone acetylation in vitro. HAT assay with HeLa nuclear extract in the presence of increasing concentrations of carnosol. Values represented in percent were calculated from three independent experiments carried out in triplicate and are represented as mean ± SEM. (**p < 0.005, ***p < 0.001). (B–D) Carnosol inhibits P300 (B) but not PCAF (C) or GCN5 (D). HAT assay was performed with p300 catalytic domain (aa 1,284–1,673), recombinant PCAF and recombinant GCN5, recombinant H3 with or without increasing carnosol. HAT activity was measured by Western Blotting scoring for acetylated histone H3. (E) HAT assay was performed with recombinant p300 catalytic domain (aa 1,284–1,673) and core histone with or without carnosol and HAT activity was measured as in (B). Data shown for all HAT assays are representative of three independent experiments. The intensities of the bands were quantified using the ImageJ software (National Institute of Health, USA).
Fig 4: Comparison of H3 N-tail signals among the chromatosome, nucleosome, and NCP(A) Superposition of 1H–15N HSQC spectra of the H3 N-tail in the NCP (blue), nucleosome (red), and chromatosome (i.e., nucleosome bound to H1.4; black) at 25 mM NaCl.(B) Superposition of 1H–15N HSQC expanded spectra of the H3 N-tail in the NCP at 100 mM NaCl (blue), nucleosome at 25 mM NaCl (red), and chromatosome (black) at 25 mM NaCl. Shown are the expanded spectra of 14 residues whose signals were shifted by the binding of H1.4.(C) Expanded spectra of 10 residues with two signals due to binding of H1.4. Symbols indicate the NCP side of doublet signal (filled diamond) and nucleosome side of doublet signal (filled hexagon).(D) DNA interaction model of the H3 N-tail in the chromatosome. The BS22 region of the H3 N-tails adopts an NCP-like form, while the BS1, L1, and BS21 regions of H3 N-tails adopt two conformations: an NCP-like form and a nucleosome-like form (PDB: 7K5Y). The positions of BS22 regions are likely to correspond to the H3 N-tail densities observed in the cryo-EM structure (Zhou et al., 2021).(E) Signal changes of K14 and acetylated K14 of the H3 N-tail in the nucleosome (red) and chromatosome (black) after the addition of Gcn5.(F) Comparison of K14 acetylation rate in the nucleosome (red circle) and chromatosome (black circle) by time-resolved NMR spectroscopy. Signal intensities were normalized by the initial signal measured before the addition of Gcn5. Red and black lines represent the data fit to the exponential equation for the nucleosome and chromatosome, respectively.See also Figure S4.
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