To our best knowledge the resulting dataset represents the most complete representation of histone H3 variant sequences available.
Simultaneous quantitative measurement of all of these tryptic peptides allowed us to monitor overall nuclear representation of histone H2A family members, H2A.X, and, importantly, allowed us to estimate the rate of phosphorylation of H2A.X histone by monitoring γH2A.X peptide in the tested samples.
A heat map representation of various histone modifications suggests that active histone modifications (H3K4me2, H3K4me3, H3K9ac and H3K14ac) are grouped together indicating a cumulative action presumably for efficient gene expression (Fig. 1c, green square).
Under-representation of repressive histone marks could be indicative of epigenetic plasticity in stem cells [7].
In other cell types epigenetic plasticity is associated with under-representation of repressive histone marks [6], [7].
Prior to decline in the nuclear H2A protein levels in senescent cells, there was a statistically significant increase (p-value= 5×10−10) in the representation of these histones within the nuclei of the HCA2 cells during acute DDR (3 h after the addition of bleomycin to the media).
Fig. 1 Schematic representation of reversible modifications of histone H3K4, K9, and K27.
In the present study, using ChIP, an in situ technique that offers a more physiological representation of nuclear events, we studied the association of histone methylation with specific DNA sequences, to further understand the role of histone H3K9me2 in transcription.
Graphical representation of upregulated and downregulated genes was performed in R. Chromatin immunoprecipitation of histone modifications were performed according to Lee et al. (2006) with minor modifications, in biological duplicate for H3K27ac, Suz12 and FLAG.
While we observed an initial increase (about 36%) in the nuclear abundance of histone H2A family members, our data clearly depict a second trend: Histone H2A family nuclear representation dropped by 70%, when compared with the stage of acute DNA damage and 60%, when compared with the initial amount of histones H2A in normally proliferating HCA2 cells.
