Histone H4 proteoforms and post-translational modifications in the Mus musculus brain with quantitative comparison of ages and brain regions.

Histone proteins are essential to the regulation of the eukaryotic genome. Histone post-translational modifications (PTMs) and single-molecule combinations of these modifications (proteoforms) allow for the regulation of many DNA-templated processes, most notably transcription. Histone H4 is a part of the core histone octamer, which packages DNA into nucleosomes. Top-down proteomics allows for the inquiry of the epigenetic landscape with proteoform-level specificity. Although these approaches are well-demonstrated ex vivo, our knowledge of in vivo histone proteoform biology remains sparse. Here, we demonstrate the first in vivo quantitative top-down analysis of histone H4 and analyze the forebrains and hindbrains of differently aged mice. This reveals novel differences between the mouse forebrain and hindbrain and region-specific changes during adolescence in histone H4 PTMs and proteoforms. At 25 days of age (P25), histone H4 of the hindbrain is more acetylated than the forebrain. At 47 days of age (P47), there are fewer significant differences in histone H4 PTMs and their combinations between regions. Histone H4 of the forebrain is more acetylated in P47 than in P25 forebrains. Hindbrains exhibit the opposite difference with histone H4 of the P25 hindbrain being more acetylated than that of P47 hindbrains. These differences are mainly driven by less abundant hyperacetylated proteoforms. Transcription of histone acetyltransferases such as p300, CBP, and HAT1 is known to be higher in cortical neurons, consistent with the observed acetylation levels. Lysine 20 methylation (K20me1, K20me2, and K20me3) is notably invariant with brain region and age difference.