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. 2013 Oct;8(10):1101-13.
doi: 10.4161/epi.26025. Epub 2013 Aug 15.

Initial characterization of histone H3 serine 10 O-acetylation

Laura-Mae P Britton  1 , Alyshia Newhart  2 , Natarajan V Bhanu  3 , Rupa Sridharan  4 , Michelle Gonzales-Cope  1 , Kathrin Plath  5 , Susan M Janicki  2 , Benjamin A Garcia  3
Affiliations

Initial characterization of histone H3 serine 10 O-acetylation

Laura-Mae P Britton et al. Epigenetics. 2013 Oct.

Abstract

In eukaryotic organisms, histone posttranslational modifications (PTMs) are indispensable for their role in maintaining cellular physiology, often through their mediation of chromatin-related processes such as transcription. Targeted investigations of this ever expanding network of chemical moieties continue to reveal genetic, biochemical, and cellular nuances of this complex landscape. In this study, we present our findings on a novel class of histone PTMs: Serine, Threonine, and Tyrosine O-acetylation. We have combined highly sensitive nano-LC-MS/MS experiments and immunodetection assays to identify and validate these unique marks found only on histone H3. Mass spectrometry experiments have determined that several of these O-acetylation marks are conserved in many species, ranging from yeast to human. Additionally, our investigations reveal that histone H3 serine 10 acetylation (H3S10ac) is potentially linked to cell cycle progression and cellular pluripotency. Here, we provide a glimpse into the functional implications of this H3-specific histone mark, which may be of high value for further studies of chromatin.

Keywords: chromatin; epigenetics; histone; mass spectrometry; post-translational modifications; proteomics; quantitative; stem cells.

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Figures

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Figure 1. Sequencing of H3S10ac peptide. (Left) MS/MS spectrum of the [M+2H]2+ ion at 556.309 m/z, which was generated from the CAD fragmentation of the prKprS(OAc)TGGKprAPR precursor peptide. Expected mono-isotopic b- (top row) and y-type (bottom row) ion fragment masses are observed. The observed accurate mass, [M+2H]2+(exp), corresponded precisely to the calculated accurate mass, [M+2H]2+(cal) of 556.309 m/z (0 ppm error). OAc refers to the acetyl modification on the S10 residue and pr refers to the propionyl amide group from chemical derivatization of histone proteins. (Right) Structure of an acetylated serine residue
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Figure 2. Yeast S22ac and Drosophila T22ac. The top panel shows the MS/MS spectrum of the [M+2H]2+ precursor ion at 591.845 m/z, the 18–26 peptide (prKprQLAS[OAc]KprAAR) of histone H3 of S. cerevisiae. Conversely, the bottom panel shows the MS/MS spectrum of the complementary 18–26 precursor peptide, extracted from Drosophila S2 cell histone samples. Both spectra show that the residue at position 22 is acetylated in a manner that is independent of the residue; serine in yeast and threonine in Drosophila.
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Figure 3. Extracted ion chromatogram for peptide quantification. The left panel shows extracted ion chromatograms for various modified peptides ([M+2H]2+ ions) spanning the H3 9–17 residues, KSTGGKAPR, after chemical derivatization by propionylation. Labels indicate the particular modified form eluted in that peak, as determined after inspection of the corresponding MS/MS spectra. Occasionally, non-target peptide peaks with identical precursor masses will elute slightly after the S10ac peptide. For example, the right panel shows a detailed MS/MS analysis of a prominent peptide peak (H3S10prK14ac) with an identical [M+2H]2+ value of 556.309 m/z, which eluted within 45 s of the targeted H3S10ac peptide. The S10pr and S10ac peptide species contain the same modifications in different sequences (S10pr - prKprSprTGGKacAPR, S10ac - prKprS[OAc]TGGKprAPR) and therefore the same m/z values and elute at almost identical times. For quantification, the relative abundance of each peak, expressed as percentages, is calculated by measuring the area under the XIC peak corresponding to each specifically modified form and expressing that value as a fraction of the total sum of the peak areas corresponding to all observed modified forms.
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Figure 4. Detection and validation of H3S10ac. A comparison of the MS/MS spectra of the [M+2H]2+ ion at 556.309, prKprS(OAc)TGGKprAPR for the in vivo-derived (top panel) and synthetic (bottom panel) H3 9–17 peptides.
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Figure 5. Immunoaffinity competition assay. (A) Validation of α-H3S10ac specificity: increasing concentrations (2–50µg) of differently modified peptides were blotted on to a nitrocellulose membrane and incubated with purified polyclonal α-H3S10ac. The antibody recognized only the synthetic H3S10ac peptide. (B) Bulk iPSC histones were analyzed by western blot for detection of H3S10ac. The signal was competed away by pre-incubation of the synthetic H3S10ac peptide.
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Figure 6. Confocal microscopy imaging of H3S10ac. Confocal images of U2OS (AandB), HeLa (CandD), and ESCs (EandF) stained with α-H3S10A antibody and DAPI to denote the nucleus. Maximum projections are shown. Scale bar in panel f represents 5 µm.
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Figure 7. Quantification of H3S10ac during reprogramming. Differentiated somatic MEFs were reprogrammed to iPSCs over the course of 21 d by overexpressing the four (4) Yamanaka transcription factors, Oct4, c-Myc, Sox2, Klf4. The levels of H3S10ac were quantified during this time course by nano-LC-MS analysis. Three independent experiments were performed and the error bars represent standard error.
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Figure 8. H3S10ac through HeLa cell cycle. HeLa suspension cells were synchronized at M phase of cell cycle through a thymidine-nocodazole synchronization followed by release into nocodazole-free media. The levels of H3S10ac were quantified at 6, 8, 10, and 12-h time points after release through nano-LC-MS analysis. Three independent experiments were performed and the error bars represent standard error.
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