Wakamori M et al. (2020), Quantification of the effect of site-specific h...

XB-ART-57561

Nucleic Acids Res 2020 Dec 16;4822:12648-12659. doi: 10.1093/nar/gkaa1050.

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Quantification of the effect of site-specific histone acetylation on chromatin transcription rate.

Wakamori M , Okabe K , Ura K , Funatsu T , Takinoue M , Umehara T .

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Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ∼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.

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Species referenced: Xenopus laevis
Genes referenced: fos usp9x
GO keywords: histone acetylation [+]

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	Figure 1. Reconstitution of H4-tetra-acetylated di-nucleosomes for chromatin transcription. (A) Scheme of di-nucleosome rRNA gene cassettes. Internal control regions (box A; IE, intermediate element; box C) of the 5S rRNA gene are indicated. The X5S 197-2 construct (27) (top) was modified by introducing a c-fos antisense probe sequence (bottom) for fluorescence correlation spectroscopy measurements. (B) Western blotting of unmodified and site-specifically tetra-acetylated histone H4 proteins. Kac, acetylated lysine. (C, D) Atomic force microscopy images of 5S rRNA gene di-nucleosomes reconstituted with unmodified histone H4 (C) or K5/K8/K12/K16-tetra-acetylated H4 (D). (E) Di-nucleosome digestion with micrococcal nuclease (MNase). Lanes 1, 3, 5, 7 and 9, di-nucleosomes with unmodified H4; lanes 2, 4, 6, 8 and 10, di-nucleosomes with K5/K8/K12/K16-tetra-acetylated H4. Lanes 1 and 2, di-nucleosomes were incubated in MNase reaction buffer in the absence of MNase. Units of MNase (Takara, cat. #2910A) per microgram DNA: lanes 3 and 4, 2.5; lanes 5 and 6, 5.0; lanes 7 and 8, 10, lanes 9 and 10, 20. (F) Agarose gel electrophoreses of di-nucleosomes constructed with c-fos-derived annealing sequence DNA. Lane M, DNA ladder marker (NEB, cat. N3232S); lane 1, di-nucleosome reconstituted with unmodified H4; lane 2, di-nucleosome reconstituted with K5/K8/K12/K16-tetra-acetylated H4.
	Figure 2. Real-time detection of chromatin transcription by fluorescence correlation spectroscopy. (A) Scheme of a Cy3-labeled antisense 2′-O-methyl RNA (2OMe-RNA) probe and its hybridization with RNA. (B) Scheme of the setup of fluorescence correlation spectroscopy (FCS) for monitoring RNA synthesis. Changes in diffusion of the antisense probe upon hybridization with transcripts in the confocal volume were detected by an avalanche photodiode (APD) at the single-molecule level. (C) Fluorescence autocorrelation functions [FAF, G(τ)] in reaction solutions. FAF of the antisense probe with RNA showed longer correlation time than that without RNA or that of a control probe with RNA. Circles, data points; solid lines, fitting curves. (Inset) Averaged diffusion time of antisense probes. Mean ± standard deviation (N = 3). (D) Calibration of the averaged diffusion time of the antisense probe molecules as a function of their concentration. Mean ± standard deviation (N = 3). R2, coefficient of determination. In (C) and (D), end-initiated RNA was transcribed by T7 RNA polymerase from two tandem copies of the 5S rRNA gene (Figure 2A) and added to a Xenopus oocyte nuclear extract without template DNA. (E) Real-time detection of nascent 5S rRNA transcripts. Template DNAs used are shown in: gray, naked 5S rRNA gene without c-fos sequence; black, naked 5S rRNA gene containing the c-fos-derived annealing sequence; red, H4-tetra-acetylated di-nucleosome 5S rRNA gene containing c-fos sequence; and blue, non-acetylated di-nucleosome 5S rRNA gene containing c-fos sequence. Mean ± standard error (N ≥ 3).
	Figure 3. Schematic diagram of 5S rRNA chromatin transcription and its real-time detection.
	Figure 4. Kinetic model of chromatin transcription. (A) Explanation of modeling of chromatin transcription. Variables are defined in Supplementary Table S1. (B, C) Explanation of the obtained equations. (B) Transcription from chromatin (equation [5]). (C) Transcription from naked DNA (equation [6]). (D, E) Numerical simulation of chromatin transcription with different k4Kac. k4Kac (D) and different α4Kac. α4Kac (E). γ=0.01γ =0.01 nM min−1 and kp=1/15≈0.0667 kp=1/15≈0.0667 min−1 are fixed. Zξ. Zξ is the concentration of transcribed RNA, where ξ ξ is ‘4Kac’ or ‘naked’. For ‘4Kac’, equation [5] is used, where k4Kac k4Kac is changed with α4Kac= αnaked= 1α4Kac= αnaked =1 fixed (D) or α4Kac α4Kac is changed with αnaked =1αnaked=1 and k4Kac=0.1k4Kac= 0.1 min−1 fixed (E). For ‘naked’, equation [6] is used.
	Figure 5. Results of fitting to the kinetic model. (A) Determination of γ and kp. First, the γ value was determined by fitting the linear region (12–17 min) of the experimental data for the naked DNA (+ c-fos) to the kinetic model \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${Z_{{\rm{naked}}}} = \;\gamma t + {z_1}$\end{document}, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${z_1}$\end{document} is the intercept; this equation is the long-term limit of equation [6]. The fitted values were: γ = 0.052 ± 0.003 nM min−1 (‘fitting value’ ± ‘fitting error’); z1 = –0.23 ± 0.05 nM; coefficient of determination R2 = 0.99. Then, using the obtained γ, the kp value was determined by fitting the whole region (0–17 min) of the data to the kinetic model \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${Z_{{\rm{naked}}}} = \;\gamma [ {\;t\; - ( {1 - {e^{ - {k_{\rm{p}}}t}}} )/{k_{\rm{p}}}\;} ] + {z_2}$\end{document}, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${z_2}$\end{document} is the intercept introduced for resolving experimental error at the initial stage (equation [6]). The fitted values were: kp = 0.22 ± 0.01 min−1; z2 = –0.010 ± 0.01 nM; R2 = 0.99). (B) Fitting results for each condition. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${Z_\xi }$\end{document} is the concentration of transcribed RNA, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\xi$\end{document} is ‘4Kac’, ‘unmod’, or ‘naked’ (equations [5] and [6]). γ = 0.052 nM min−1 and kp = 0.22 min−1, which were obtained in (A), were used for fitting. (C) Schematic illustration of acceleration of chromatin transcription by histone H4 acetylation. k4Kac ≈ 0.15 ± 0.009 min−1 and kunmod ≈ 0.052 ± 0.006 min−1.

References [+] :

ALLFREY, ACETYLATION AND METHYLATION OF HISTONES AND THEIR POSSIBLE ROLE IN THE REGULATION OF RNA SYNTHESIS. 1964, Pubmed