Figure 8. Mouse H2A.J accumulates with age in an organ-specific fashion.

(a) H2A.J is present at low levels in proliferating mouse embryonic fibroblasts (MEFs) and increases greatly in replicatively senescent MEFs (d-SenRep) or MEFs maintained in etoposide-induced senescence for 20 days (d-SenETO). (b) Experimental timeline for the extraction of histones from the indicated C57BL/6 mouse organs. (c) Top-down deconvoluted mass spectra of intact histone H2A2 proteins extracted from mouse organs at the indicated ages. H2A.J is shown in orange and canonical H2A types 1 and 3 are shown in shades of grey. See Supplementary Table 2 for the observed and theoretical masses of these peaks and Supplementary Fig. 11 for their sequence features. (d) Relative abundance of H2A.J as the percent of all canonical H2A species by quantifying the Gly5-Arg12 tryptic peptide from H2A.J versus the same peptide from all other canonical H2A species (mean and s.d. for two biological replicates). (e) H2A.J and H3 Western blot of acid-extracted histones from the indicated mouse organs. (f) qRT–PCR analysis of H2AFJ (coding for H2A.J), HIST3H2A (coding for H2A type 3), and HIST1H2AB (coding for H2A type 1) mRNA levels in 1 year old mouse organs (three technical replicates). Random primers were used to prepare cDNA to quantify total RNA levels (left panel). The right panel shows the quantification of H2A.J, H2A type 3, and H2A type 1 proteins by top-down mass spectrometry from two biological replicates (mean and s.d.). The levels of H2A mRNA and proteins in 1-year-old mouse organs were normalized to the levels found in the brain.