Partial Replacement of Nucleosomal DNA with Human FACT Induces Dynamic Exposure and Acetylation of Histone H3 N-Terminal Tails

Abstract

The FACT (facilitates chromatin transcription) complex, comprising SPT16 and SSRP1, conducts structural alterations during nucleosome unwrapping. Our previous cryoelectron microscopic (cryo-EM) analysis revealed the first intermediate structure of an unwrapped nucleosome with human FACT, in which 112-bp DNA and the phosphorylated intrinsically disordered (pAID) segment of SPT16 jointly wrapped around the histone core instead of 145-bp DNA. Using NMR, here we clarified that the histone H3 N-terminal tails, unobserved in the cryo-EM structure, adopt two different conformations reflecting their asymmetric locations at entry/exit sites: one corresponds to the original nucleosome site buried in two DNA gyres (DNA side), whereas the other, comprising pAID and DNA, is more exposed to the solvent (pAID side). NMR real-time monitoring showed that H3 acetylation is faster on the pAID side than on the DNA side. Our findings highlight that accessible conformations of H3 tails are created by the replacement of nucleosomal DNA with pAID.

Keywords: Biochemistry; Molecular Biology; Structural Biology.

Graphical abstract

Figure 1

Structural Location of the Two H3 N-Tails in the 112-bp DNA/pAID Nucleosome (A) Domain organization of human FACT. Full-length FACT and pAID (red) proteins were used in this study. (B) Cryo-EM structure of the 112-bp DNA/pAID nucleosome. Three views of the EM density map (EMD-9639) are superimposed on the nucleosome structure (PDB: 2CV5) lacking the 33-bp DNA. Visualization of the EM maps and fitting of nucleosome structures into the maps were performed using UCSF Chimera (Pettersen et al., 2004). pAID is shown in a deep pink density. H2A, H2B, H3, H4, and DNA are colored yellow, red, blue, green, and orchid, respectively. Blue circular chains denote the H3 N-tails, which were unobserved in the EM structure. The sequence of the H3 N-tail is shown above. Superhelix locations (SHL), which represent the number of double turns from the dyad axis of the canonical nucleosome (0), are indicated.

Figure 2

Two Separate NMR Signals from H3 N-Tails in the 112-bp DNA/pAID Nucleosome (A) Backbone resonance assignments of the 112-bp DNA/pAID nucleosome in the 2D ¹H-¹⁵N HSQC spectrum. Cartoon model of the nucleosomal complex is shown below. The histone proteins and DNA are colored as follows: H2B (red), H2A (yellow), H3 (blue), H4 (green), and DNA (light sea green). The H3 N-tails are indicated by dotted strings. pAID is colored magenta. Red open circles labeled with P indicate phosphorylation. (B) Spectral comparison of 2D ¹H-¹⁵N HSQC spectra between the 145-bp nucleosome (red) and the 112-bp DNA/pAID nucleosome (blue). Signal assignments in the 145-bp nucleosome are labeled. Cartoon model of the nucleosomal complex is below. See also Figure S1.

Figure 3

The Two H3 Tails Adopt Distinct Conformations in the 112-bp DNA/pAID Nucleosome Two expanded regions of the spectrum of Figure 1B are separately shown (A and B). Spectral comparison of 2D ¹H-¹⁵N HSQC spectra between the 145-bp nucleosome at 200 mM NaCl (yellow) and the 112-bp DNA/pAID nucleosome (blue). As described in the text, the two separate signals from each H3 N-tail residue in the 112-bp DNA/pAID nucleosome are assigned to H3 N-tails on the pAID side (labeled by subscript p) and the DNA side (labeled by subscript D). See also Figure S2.

Figure 4

Chemical Shift Differences and Heteronuclear NOE Values of the H3 N-Tails in the Nucleosomal Complexes (A and B) Histograms showing H3 N-tail chemical shift differences between the pAID and DNA sides in the 112-bp DNA/pAID nucleosome (A) and between 0 and 200 mM NaCl conditions for the 145-bp nucleosome (B). Chemical shift differences are plotted against the residue number of the H3 N-tails. Asterisks (∗) indicate residues that were either not observed or not assigned. The dagger (†) indicates Arg2, for which the chemical shift difference was calculated for two separate signals that were not correctly assigned to either the pAID or DNA side H3 N-tails. (C and D) Profiles of the ¹⁵N{¹H} heteronuclear NOE values of H3 N-tails in the 112-bp DNA/pAID nucleosome (C) and the 145-bp nucleosome (D). NOE values are plotted against the residue numbers of the H3 N-tails. Asterisks (∗) indicate residues that were either not observed or not assigned. The pAID and DNA side signals in the 112-bp DNA/pAID nucleosome are shown in blue and red, respectively. The dagger (†) indicates Arg2, for which the value of the right signal (R2 in Figure S2D) is nominally shown in red and the value of the left signal (R2∗ in Figure S2D) is shown in blue (112-bp DNA/pAID nucleosome) or gray (145-bp nucleosome). The double dagger (‡) indicates Arg7, for which the signals on the DNA side and in the 145-bp nucleosome were not correctly calculated owing to severe overlap with Lys36 signals, as shown in Figures 2A and 2B. Error bars were calculated on the basis of the signal to noise ratio.

Figure 5

Comparison of H3 K14 Acetylation between the Canonical Nucleosome and Nucleosomal Complexes with hFACT Proteins (A–C) Expanded view of the ¹H-¹⁵N NMR spectrum at Lys14 of the H3 N-tail in the 112-bp DNA/pAID nucleosome (A), the 145-bp nucleosome (B), and the 145-bp nucleosome at 200 mM NaCl (C) during acetylation by Gcn5. (D) Time-resolved NMR profiling of consecutive enzymatic reactions. Red, blue, black, and yellow plots show the raw values of the signal intensity ratio at Lys14 on the DNA and pAID sides in the 112-bp DNA/pAID nucleosome and in the 145-bp nucleosome at 0 and 200 mM NaCl, respectively. Values were calculated by setting the signal intensity before the reaction or after 1 h (200 mM NaCl) as 1. The decay curves were fitted with first-order equations using Gnuplot. (E) Fluorescence HAT assays of the indicated nucleosomal complexes with hFACT proteins. HAT activity was quantified by the fluorescence intensity measured after the reaction. Averages from at least three independent experiments are shown; error bars represent SD. Addition of hFACT causes unexpected background, probably because Gcn5 may partly acetylate lysine residues within hFACT. See also Figure S3.

Figure 6

Conformational Comparison between the pAID- or DNA-Bound H3 Peptide and the Two H3 N-Tails in the 112-bp DNA/pAID Nucleosome Two spectral regions are separately shown (A and B). Spectral comparison of 2D ¹H-¹⁵N HSQC spectra between the H3 peptide upon the 10-fold addition of DNA (red) or pAID (cyan) and the 112-bp DNA/pAID nucleosome (black). Arrows connect the pAID side signal to the DNA side signal in the 112-bp DNA/pAID nucleosome. The double dagger (‡) indicates Lys36, which was followed by an additional tryptophan residue in the H3 peptide (differing from Lys37 in the native sequence of H3), and for which the chemical shift values could not be correctly compared with those in the 112-bp DNA/pAID nucleosome. (C) Expanded regions of the superposition are shown for the comparison of selected residues. (D) Chemical shift differences (Δδ) between the pAID side H3 N-tail and the DNA side H3 N-tail (black) or the H3 peptide upon 10-fold DNA (red) or pAID (cyan) addition. Chemical shift differences are plotted against residue numbers of H3 N-tails. Asterisks (∗) indicate residues that were either not observed or not assigned. The dagger (†) indicates Arg2, for which the chemical shift differences were calculated by using the nominal definition of the right and left signals in Figure S2D as the DNA and the pAID sides, respectively. The double dagger (‡) indicates Lys36, which was followed by an additional tryptophan residue in the H3 peptide (differing from Lys37 in the native sequence of H3), and for which the chemical shift values could not be correctly compared with those in the 112-bp DNA/pAID nucleosome. See also Figures S4–S6.

Figure 7

Summary of the Dynamic Conformations of the Nucleosomal H3 N-Tails (A) Dynamic structures of two H3 N-tails in the 112-bp DNA/pAID nucleosome. Two views of the EM density map (EMD-9639) on the nucleosome structure (PDB: 2CV5), colored as in Figure 1B, are shown. Blue circular chains denote the H3 N-tails. Dotted arrows represent the dynamic behavior of the H3 N-tails. The dashed ellipsoid, shaded in red, represents the space between two DNA gyres within the canonical nucleosome or on the DNA side in the 112-bp DNA/pAID nucleosome. (B) DNA-binding model of H3 N-tails in the canonical nucleosome. A side view of the nucleosome structure (PDB: 2CV5), colored as in Figure 1B, is shown. Blue ellipsoids represent the two basic segments, B1 and B2, in the H3 N-tail. Orange strings represent the linkers, L1 and L2, in the H3 N-tail. Dotted arrows represent the dynamic behavior of the H3 N-tails. See also Figure S7.