Quantitative middle-down MS analysis of histone H3 proteoforms throughout lifespan in mice

Replication-independent deposition of histone variant H3.3 into chromatin is essential for many biological processes, including development, oogenesis and nuclear reprogramming. 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. To address this, we investigated genome-wide changes in histone H3 variants composition and H3 modification abundances throughout the lifespan in mice using quantitative mass spectrometry (MS) – based middle-down proteomics strategy. Using middle-down MS we demonstrate that H3.3 accumulates in the chromatin of various somatic mouse tissues throughout life, resulting in near complete replacement of H3.1/2 isoforms by the late adulthood. Accumulation of H3.3 is associated with profound changes in the global level of H3 methylation. H3.3-containing chromatin exhibits distinct stable levels of H3R17me2 and H3K36me2, different from those on H3.1/H3.2-containing chromatin, indicating a direct link between H3 variant exchange and histone methylation dynamics with age. In summary, our study provides the first time comprehensive characterization of dynamic changes in the H3 modification landscape during mouse lifespan and links these changes to the age-dependent accumulation of histone variant H3.3.

组蛋白变体H3.3（histone variant H3.3）不依赖复制的染色质沉积，对包括发育、卵子发生和细胞核重编程在内的诸多生物学过程至关重要。与依赖复制的H3.1/2亚型不同，H3.3在整个细胞周期中均有表达，且随年龄增长在有丝分裂后细胞中富集。然而，H3变体替换的终生动态变化及其对染色质组织的影响，目前仍未完全明确。为解决这一科学问题，本研究采用基于定量质谱（MS）的中下行蛋白质组学（middle-down proteomics）策略，对小鼠整个生命周期内的组蛋白H3变体组成及H3修饰丰度的全基因组变化进行了分析。通过中下行质谱分析，本研究证实H3.3在小鼠多种体细胞组织的染色质中随生命周期不断积累，至成年晚期时几乎完全替换了H3.1/2亚型。H3.3的积累与组蛋白H3甲基化水平的全局性显著变化密切相关。携带H3.3的染色质其H3R17me2和H3K36me2修饰水平稳定且具有特异性，与携带H3.1/H3.2的染色质上的修饰水平存在显著差异，这表明H3变体替换与年龄相关的组蛋白甲基化动态变化之间存在直接关联。综上，本研究首次全面表征了小鼠生命周期内H3修饰谱的动态变化，并将这些变化与年龄依赖性的组蛋白变体H3.3积累建立了关联。