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. 2009 Oct 9;36(1):153-63.
doi: 10.1016/j.molcel.2009.07.027.

A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation

Heinz Neumann  1 , Susan M Hancock, Ruth Buning, Andrew Routh, Lynda Chapman, Joanna Somers, Tom Owen-Hughes, John van Noort, Daniela Rhodes, Jason W Chin
Affiliations

A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation

Heinz Neumann et al. Mol Cell. .

Abstract

Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.

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Figures

Figure 1
Figure 1
Selection of an Improved Acetyl-Lysyl-tRNA Synthetase/tRNACUA Pair for the Incorporation of Acetyl-Lysine in Recombinant Proteins (A) The active site of M. mazei PylRS bound to pyrrolysine (figure created using PyMOL [http://www.pymol.org] and PDB file 2Q7H). The residues mutated relative to the wild-type sequence are shown as sticks. Residues in cyan are mutated in the progenitor AcKRS-1 and were randomized again in the new library; A267 (magenta) was only included in the new library. (B) Characterization of a more efficient acetyl-lysyl-tRNA synthetase/tRNACUA pair. Myoglobin-His6 was expressed in E. coli DH10B from pMyo4TAG PylT (Neumann et al., 2008) (containing a hexa-histidine-tagged myoglobin gene with an amber codon at position 4 and the gene encoding MbtRNACUA) in the presence or absence of 10 mM acetyl-lysine using either pBK AcKRS-1 or pBK AcKRS-3. The proteins were purified by Ni2+ chromatography and analyzed by 4%–12% SDS-PAGE or detected in total lysates by western blot with an anti-His6 antibody.
Figure 2
Figure 2
The Expression and Purification of Site-Specifically Acetylated Histones and the Assembly of Histone Octamers and Nucleosomes (A) Schematic illustration showing the recombinant expression of site-specifically acetylated recombinant histones in E. coli and their reconstitution into histone octamers and nucleosomes. (B) (Left) The expression, purification, and TEV cleavage of histone H3 K14Ac is followed by SDS PAGE. (Right) Purified and TEV cleaved site-specifically acetylated histones. (1) molecular weight marker, (2) H3 WT, (3) H3 K14Ac, (4) H3 K23Ac, (5) H3 K27Ac, (6) H3 K56Ac, (7) H2A WT, (8) H2A K9Ac, (9) H2B WT, (10) H2B K5Ac, and (11) H2B K20Ac. (C) Electrospray ionization mass spectrometry demonstrates that the protein is homogeneously acetylated, and MS/MS of tryptic peptides identifies the site of acetylation at lysine 56, as genetically encoded. The smaller peak to the right of the main peak is 98 Da heavier and corresponds to a phosphate from buffer associated with the histone. (D) H3 K56Ac assembles into octamers with comparable efficiency to unmodified H3. Denaturing (4%–12%) SDS-PAGE of assembled octamers. The acetylation of H3 in the octamer is confirmed by western blot with an anti-acetyl-lysine antibody. (E) Reconstitution of unmodified octamers and octamers bearing H3 K56Ac into nucleosomes with 197bp 601 DNA. Nucleosomes and free DNA were resolved by 0.8% agarose gel and stained with ethidium bromide.
Figure 3
Figure 3
Nucleosome Stability and Dynamic Partial Unwrapping of Nucleosomal DNA Measured by FRET Using Three-Way Labeled Nucleosomes (A) Schematic of the nucleosome highlighting the locations of the fluorescence donor Cy3 (green) at the 5′ end of the DNA, the acceptor dye Cy5 (red) coupled to histone H2A K119C, and the site of acetylation, H3 K56Ac (blue). The figure was created using the PDB file 1KX5 and PyMOL (http://www.pymol.org). (B) Analysis of nucleosome reconstitution by 0.8% agarose gel electrophoresis, in which lane 1 = 100 bp DNA ladder, lane 2 = naked Cy3-labeled DNA, lane 3 = Cy5-labeled H2A K119C nucleosome reconstitution with wild-type H3, and lane 4 = Cy5-labeled H2A K119C nucleosome reconstitution with H3 K56Ac. (C) The salt-induced dissociation of nucleosome core particles can be monitored by FRET. (Left) Increasing [NaCl] from 0 (red) to 1.75 M (violet) leads to decreased FRET emission from Cy5 and increased Cy3 emission (arrows). Excitation wavelength was set at 515 nm. (Right) Equilibrium dissociation curves were obtained by monitoring changes in fluorescence donor and acceptor emission at 565 and 670 nm, respectively. Data were normalized using the upper and lower plateau values as baselines, with wild-type nucleosomes in orange and H3 K56Ac nucleosomes in magenta. The data represent the mean values, and the error bars represent ± 1 SD.
Figure 4
Figure 4
spFRET Experiments on Transient Unwrapping of DNA and DNA Breathing Demonstrate that K56 Acetylation Promotes Local Unwrapping Near the Entry-Exit Points of the Nucleosome (A) Schematic of the labeling positions on the nucleosome DNA. The end label fluorophore pair (Cy3B/Atto647N) is close to the entry-exit point of the nucleosome at position −2, and the internal label pair is at −27 from the entry-exit point. The position of K56 is shown in blue. The figure was created using the PDB file 1KX5 and PyMOL (http://www.pymol.org). (B and C) spFRET efficiency measured for nucleosomes reconstituted with internally or end-labeled DNA, respectively, using a combination of native PAGE, ALEX, and FCS as described in the Experimental Procedures.
Figure 5
Figure 5
Assembly and Sedimentation Analysis of Nucleosome Arrays Bearing Homogeneously Acetylated Nucleosomes (A) Titration of purified histone H3 K56Ac octamers to assemble chromatin arrays containing 61 repeats of 197 bp of the 601 nucleosome-positioning DNA sequence. A retarded gel shift indicates loading of the DNA array with histone octamers. Excess histone octamer forms nucleosome core particles (NCPs) with competitor DNA (crDNA). Conditions of lane 4 were used to reconstitute DNA arrays in subsequent experiments. (B) DNA arrays were reconstituted with saturating amounts of histone octamer and with increasing amounts of H5 linker histone in order to induce compaction. Chromatin arrays were folded in 1 mM MgCl2 and 10 mM TEA (pH 7.4), and the degree of the compaction was measured quantitatively by sedimentation velocity analysis.
Figure 6
Figure 6
H3 K56 Acetylated Nucleosomes Cause Minimal Alteration to the Initial Rate of RSC or SWI/SNF Repositioning (A and B) Competitive repositioning assays were performed using 1 pmol each of H3 K56Ac and wild-type nucleosomes, 1 mM of ATP, and 41 fmol of RSC (A) or 115 fmol of SWI/SNF (B). A representative native PAGE gel of the repositioning assay is shown for each remodeler. The initial rate estimate for repositioning of H3 K56Ac nucleosomes relative to wild-type for RSC was 1.2 fold ± 0.1 (mean ± SE of the mean) and for SWI/SNF was 1.4 fold ± 0.2. Each experiment was repeated in triplicate. Asterisks indicate the P position. WT, wild-type. (C and D) H3 K56Ac and wild-type nucleosomes exhibit equivalent remodeler-driven dimer transfer. Remodeler dimer transfer was performed using 0.25 pmol of donor nucleosomes assembled with Cy5-labeled H2A onto 54A18 DNA fragments, 0.75 pmol of wild-type tetrasome acceptor assembled on 0W0 DNA fragments, 1 mM of ATP, and 83 fmol of RSC (C) or 230 fmol of SWI/SNF (D). For each dimer transfer experiment, a representative Cy5 scan of the native PAGE gel is shown. Both RSC and SWI/SNF caused a 1.2-fold increase of the percentage of dimer transfer for H3 K56Ac nucleosomes relative to wild-type at the finish of their respective time courses. As the SE of the mean was large in both cases, 0.1 and 0.2 for RSC and SWI/SNF, respectively, there was no significant change in the percentage of dimer transfer. Each experiment was repeated in triplicate.
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