N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

Abstract

The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N(6)-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3'-formylphosphate residues arising from 5'-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N(6)-amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N(6)-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04-0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose-response relationship for the formation of N(6)-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N(6)-formylation of the linker histone H1. The N(6)-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N(6)-acetylation recognized as an important determinant of gene expression in mammalian cells. The N(6)-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

Fig. 1.

N⁶-formyl-lysine residues arising from products of oxidative DNA damage are chemical analogs of the biologically important lysine N⁶-acetylation.

Fig. 2.

Reactive 3′-formylphosphate residues arise from 5′-oxidation of deoxyribose in DNA.

Fig. 3.

HPLC analysis of neocarzinostatin-induced ³H-labeling of histones from TK6 cells with DNA containing 5′-[³H]thymidine (Upper) or methyl-[³H]thymidine (Lower). Isolated nuclei were treated with neocarzinostatin (30 μM; shaded bars) or methanol vehicle (open bars), followed by histone extraction, nuclease digestion, and HPLC separation, as described in Materials and Methods. Tritium associated with the various histone-containing HPLC fractions was quantified by scintillation counting and reported as disintegrations per minute (DPM) per unit area of UV absorbance signal. Data represent mean ± SD for three independent experiments.

Fig. 4.

N⁶-formyl-lysine formation in TK6 nuclei (closed circles) and cells (open circles) treated with neocarzinostatin.