Comparative Proteomic Analysis of Histone Modifications upon acridone derivative 8a-Induced CCRF-CEM cells by Data Independent Acquisition.

Abstract

The lead compound acridone derivative 8a showed potent anti-proliferative activity by inducing DNA damage through direct stacking with DNA bases and triggering ROS in CCRF-CEM cells. To define the chromatin alterations during DNA damage sensing and repair, a detailed quantitative map of single and co-existing histone post-translational modifications (PTMs) in CCRF-CEM cells affected by 8a was performed by Data Independent Acquisition (DIA) method on QE-plus. A total of 79 distinct and 164 co-existing histone PTMs were quantified, of which 16 distinct histone PTMs exhibited significantly altered when comparing 8a-treated cells with vehicle control cells. The changes in histone PTMs were confirmed by western blotting analysis for four H3 and one H4 histone markers. The up-regulated di-methylation on H3K9, H3K36 and H4K20 implied that CCRF-CEM cells might accelerate DNA damage repair in order to counteract the DNA lesion induced by 8a, which was verified by an increment in the 53BP1 foci localization at the damaged DNA. Most of the significantly altered PTMs involved in transcriptional regulation, including down-regulated acetylation on H3K18, H3K27 and H3K122, and up-regulated di, tri-methylation on H3K9 and H3K27. This transcription silencing phenomenon was associated with G2/M cell cycle arrest after 8a treatment by flow cytometry. This study shows that DIA proteomics strategy provides a sensitive and accurate way to characterize the co-existing histone PTMs changes and their cross-talk in CCRF-CEM cells after 8a treatment. Specifically, histone PTMs rearrange transcription-silencing and cell cycle arrest DNA damage repair may contribute to the mechanism of epigenetic response affected by 8a.