Early butyrate induced acetylation of histone H4 is proteoform specific and linked to methylation state

Histone posttranslational modifications (PTMs) help regulate DNA templated processes; however, relatively little work has unbiasedly explored the single-molecule combinations of histone PTMs, their dynamics on short timescales, or how these preexisting histone PTMs modulate further histone modifying enzyme activity. We use quantitative top down proteomics to unbiasedly measure histone H4 proteoforms (single-molecule combinations of PTMs) upon butyrate treatment. Our results show that histone proteoforms change in cells within 10 minutes of application of sodium butyrate. Cells recover from treatment within 30 minutes after removal of butyrate. Surprisingly, K20me2 containing proteoforms are the near-exclusive substrate of histone acetyltransferases upon butyrate treatment. Single-molecule hierarchies of progressive PTMs mostly dictate the addition and removal of histone PTMs (K16ac > K12ac ≥ K8ac > K5ac, and the reverse on recovery). This reveals the underlying single-molecule mechanism that explains the previously reported but indistinct and unexplained patterns of H4 acetylation. Thus, preexisting histone PTMs strongly modulate histone modifying enzyme activity and this suggests that proteoform constrained reaction pathways are crucial mechanisms that enable the long-term stability of the cellular epigenetic state.

组蛋白翻译后修饰（posttranslational modifications, PTMs）可调控以DNA为模板的各类生物学过程；然而，目前鲜有研究能够无偏地解析组蛋白PTMs的单分子组合模式、其在短时间尺度上的动态变化，以及这些预先存在的组蛋白PTMs如何调控后续组蛋白修饰酶的活性。本研究采用定量自上而下蛋白质组学技术，无偏地检测丁酸盐处理后组蛋白H4蛋白亚型（proteoforms，即PTMs的单分子组合）的变化情况。研究结果显示，在加入丁酸钠后的10分钟内，细胞内的组蛋白蛋白亚型即发生改变；而在移除丁酸盐后的30分钟内，细胞即可恢复至处理前状态。令人意外的是，经丁酸盐处理后，携带K20me2修饰的蛋白亚型是组蛋白乙酰转移酶的近乎唯一底物。逐步发生的PTMs的单分子层级调控机制，主导了组蛋白PTMs的添加与移除过程（调控优先级为K16ac > K12ac ≥ K8ac > K5ac，细胞恢复阶段则呈现相反趋势）。该发现揭示了潜在的单分子作用机制，可解释此前已被报道但仍模糊不清、尚未得到合理解释的H4乙酰化模式。综上，预先存在的组蛋白PTMs可强烈调控组蛋白修饰酶的活性，这表明受蛋白亚型约束的反应通路是维持细胞表观遗传状态长期稳定性的关键机制。