Tvardovskiy Andrey, Wrzesinski Krzysztof, Sidoli Simone, Fey Stephen J, Rogowska-Wrzesinska Adelina, Jensen Ole N
From the ‡Department of Biochemistry and Molecular Biology and §Center for Epigenetics, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK - 5230 Odense M, Denmark.
From the ‡Department of Biochemistry and Molecular Biology and.
Mol Cell Proteomics. 2015 Dec;14(12):3142-53. doi: 10.1074/mcp.M115.048975. Epub 2015 Sep 30.
Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.
组蛋白的翻译后修饰（PTMs）在DNA模板化过程的调控中发挥着重要作用。越来越多的证据表明，组蛋白N端尾巴的蛋白水解切割，即组蛋白剪切，会影响核小体动力学和功能特性。通过质谱的自上而下和中而下蛋白质分析，我们报告了人类肝细胞中的组蛋白H2B和H3 N端尾巴剪切，并证明了剪切与组蛋白H3共存的PTMs之间的关系。当在球体（3D）培养中生长时，组蛋白H2B和H3在原代人肝细胞和肝癌细胞系HepG2/C3A中会发生蛋白水解加工，但在平板（2D）培养中则不会。利用串联质谱，我们在H3中定位了四个不同的剪切位点，在H2B中定位了一个剪切位点。我们表明，在球体培养中，剪切的H3蛋白变体主要由经典组蛋白H3代表，而在原代肝细胞中，超过90%的剪切H3对应于组蛋白变体H3.3。对组蛋白H3修饰的综合分析揭示了一系列PTMs，包括K14me1、K27me2/K27me3和K36me1/me2，它们在剪切的和完整的H3中丰度不同。对共存PTMs的分析揭示了剪切的H3中H3K36甲基化和H3K23乙酰化之间的负性串扰。我们的数据提供了人类肝细胞中组蛋白剪切的首个证据，并证明剪切的H3携带与完整H3不同的独特共存PTMs。
