Simultaneous metabolic labeling of cells with multiple amino acids: Localization and dynamics of histone acetylation...

Simultaneous metabolic labeling of cells with multiple amino acids combined with acetic acid urea-PAGE and MS was used to characterize histones in Kasumi-1 cells treated with the histone deacetylase inhibitor depsipeptide (DDP). The approach allowed for rapid targeting, identification, and subsequent characterization of peptides containing sites of acetylation or methylation. Multiple methylation sites were determined for histone H3 including: di- and tri-methylation of K9 and K27; mono- and di-methylation of K36 and K79; and mono-methylation of K37. The acetylation patterns for histones H4 and H3 were established. Quantitative analysis of the modification change after treatment with DDP was also performed and the dynamics of H4 acetylation determined. Functional analysis by RT-PCR showed that DDP unregulated p21 expression with a maximum after 18-h exposure. Chromatin immunoprecipitation experiments indicated that DDP treatment caused an accumulation of hyperacetylated histone H4 and H3 isoforms and a decrease in K9 di-methylation of H3 on the p21 promoter.