Stable Histone Adduction by 4-Oxo-2-nonenal: A Potential Link between Oxidative Stress and Epigenetics

Lipid electrophiles modify cellular targets, altering their function. Here, we describe histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells: four 4-ketoamides to Lys and three Michael adducts to His. A 4-ketoamide adduct residing at H3K27 was identified in stimulated macrophages. Modification of histones H3 and H4 prevented nucleosome assembly.

In-gel digestion of histones. 2.5µg of chromatin was separated on a 15% SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA). Four gel bands were excised corresponding to each histone based on molecular weight. Gel bands were cut into 1mm 3 pieces, and treated with 45mM dithiothreitol for 30 minutes, followed by 100mM iodoacetamide for 45 minutes to alkylate all available Cys residues. 5 After reduction and alkylation, gel pieces were destained with 50% MeCN in 25mM NH 4 HCO 3. Four different digestion methods were employed to attain 100% sequence coverage over all four histones. For digestion of histones with trypsin, gel pieces were incubated with 10 ng/uL trypsin in 25mM NH 4 HCO 3 for either 16 hours (Method 1) or 3 hours (Method 2) at 37 °C. For Methods 3 and 4, gel bands were first destained and then immersed in 5.9M propionic anhydride (prepared in methanol) and incubated for 20 minutes at room temperature. 3 Propionylation was performed prior to reduction/alkylation and was repeated twice to ensure that all available lysine residues were propionylated. After each treatment with propionic anhydride, the solution was removed, gel pieces were dried under vacuum, and were subsequently rehydrated and rinsed three times with 100mM NH 4 HCO 3 . Digestion with trypsin was performed for either 16 hours (Method 3) or 1 hour (Method 4).

LC-MS/MS Analysis of Histone Modifications.
Peptides were extracted following each method using gel dehydration (60% acetonitrile, 0.1% TFA); extracts were dried via speed vacuum centrifugation, and the peptides were reconstituted in 0.1% formic acid. Peptide mixtures were loaded onto a capillary reverse-phase analytical column (360µm o.d. x 100µm i.d.) using an Eksigent NanoLC Ultra HPLC and autosampler. The analytical column was packed with 20 cm of C18 reverse-phase material (Jupiter, 3µm beads, 300 Å or Aqua C18, 3µm beads, Phenomenex), directly into a laser-pulled emitter tip. Peptides were gradient-eluted at a flow rate of 500 nL/min, and the mobile phase solvents consisted of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). A 90 min gradient was performed, consisting of the following: 0−15 min, 2% B (during sample loading); 15−60 min, 2−40% B; 60−70 min, 40−90% B; 70-73 min, 90% B; 73-76 min 90−2% B; and 76−90 min, 2% B (column re-equilibration). For select LC-MS/MS analyses, LC conditions were modified such that peptides were loaded at 0% B, and after sample loading, the peptide elution portion of the gradient consisted of 0-40% B from 15-60 min. Upon gradient elution, peptides were mass analyzed on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific), equipped with a nanoelectrospray ionization source. The instrument was operated using a data-dependent method with dynamic exclusion enabled. Full-scan (m/z 300−2000) spectra were acquired with the Orbitrap (resolution 60,000), and the top 16 most abundant ions in each MS scan were selected for fragmentation in the LTQ. An isolation width of 2 m/z, activation time of 10ms, and 35% normalized collision energy were used to generate MS2 spectra. Dynamic exclusion settings allowed for a repeat count of 2 within a repeat duration of 10 s, and the exclusion duration time was set to 15 s.
For identification of histone peptides, tandem mass spectra were searched with Sequest (Thermo Fisher Scientific) against a human subset database created from the UniprotKB protein database (www.uniprot.org). Due to the plethora of diverse histone modifications, multiple database searches were required for each sample to minimize false positives. These variable modifications included: +57.0214 (carbamidomethylation) on Cys; +15.9949 (oxidation) on Met; +14.0157 (monomethylation), +28.0313 (dimethylation), +42.0106 (acetylation) on Lys; +158.1306 (4-ONE Michael adduct) on Cys, His and Lys; +156.1150 (4-ketoamide), +138.1123 (Schiff-base 1), and +140.1279 (Schiff-base 2) on Lys. Additionally, for Methods 3 and 4, a variable mass of +56.0262 (propionylation) was applied to Lys. Search results were assembled S4 using Scaffold 3.0 (Proteome Software). Spectra of interest were inspected using Xcalibur 2.1 Qual Browser software (Thermo Scientific). Tandem mass spectra of all modified peptide precursors as well as spectra acquired of the corresponding unmodified peptide forms were examined by manual interrogation. The search parameters provided do not account for Nterminal processing and differences between trimethylation (+42.0471) and acetylation (+42.0106); therefore, all +42 mass shifts are identified as acetylation as manual validation is required for these peptides. Supplemental Table 2 contains a map for sequence coverage with the indicated method; however, only peptides containing 4-ONE modifications and gray shading were validated manually.
In vitro assembly of nucleosomes. Nucleosomes were assembled in vitro utilizing a previously developed salt dilution assay. 6 Briefly, purified H2A/H2B dimers and H3/H4 tetramers were incubated with 208bp of DNA in a solution containing 2M NaCl. The salt concentration was diluted following 5, 30-minute incubations until the NaCl concentration reached 0.25M. To assess the impact of aldehyde modification on nucleosome assembly, assembled nucleosomes, H2A/H2B dimers, or H3/H4 tetramers were treated with increasing concentrations of either a4-ONE at 37 o C for 60 minutes or 5mM acetic anhydride for 60 minutes on ice. Excess a4-ONE was quenched by the addition of 10mM N-α -Cys, whereas excess acetic anhydride was quenched with 10mM N-α -Lys. Aliquots of each sample were mixed with glycerol and resolved on a native-PAGE gel. The gel was stained in the dark with SYBR Gold for a period of 40 minutes and nucleosomes were visualized through excitation at 655nm. Acetylation was visualized via standard immunoblotting using antibodies directed against acetylated lysine (Cell Signaling, Danvers, MA). To ensure histones were present in nucleosomal bands, SYBR Gold-stained gels were then washed with ddH 2 O and stained with Coomassie blue.     Table S1. Composite list of the histone peptides identified using the methods described in Figure 2.

Table S2. A proteomic screen of chromatin reveals seven sites of modification by 4-ONE.
Chromatin extracted from RKO cells treated with a bolus dose of 4-ONE (250µM) was subjected to SDS-PAGE and in-gel digestions as described in Figure 2; sites of modification were identified using LTQ Orbitrap Velos MS. Four adducts resulted from the stable addition of a 4ketoamide adduct on a lysine residue. Sites of adduction are indicated as *;pr: propionylated lysine; species number corresponds to Figure S1.