Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape

Deposition of replication-independent histone variant H3.3 into chromatin is essential for many biological processes, including development and reproduction. Unlike replication-dependent H3.1/2 isoforms, H3.3 is expressed throughout the cell cycle and becomes enriched in postmitotic cells with age. However, lifelong dynamics of H3 variant replacement and the impact of this process on chromatin organization remain largely undefined. Using quantitative middle-down proteomics we demonstrate that H3.3 accumulates to near saturation levels in the chromatin of various mouse somatic tissues by late adulthood. Accumulation of H3.3 is associated with profound changes in global levels of both individual and combinatorial H3 methyl modifications. A subset of these modifications exhibit distinct relative abundances on H3 variants and remain stably enriched on H3.3 throughout the lifespan, suggesting a causal relationship between H3 variant replacement and age-dependent changes in H3 methylation. Furthermore, the H3.3 level is drastically reduced in human hepatocarcinoma cells as compared to nontumoral hepatocytes, suggesting the potential utility of the H3.3 relative abundance as a biomarker of abnormal cell proliferation activity. Overall, our study provides the first quantitative characterization of dynamic changes in H3 proteoforms throughout lifespan in mammals and suggests a role for H3 variant replacement in modulating H3 methylation landscape with age.