Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
J Am Soc Mass Spectrom. 2021 Jun 2;32(6):1300-1311. doi: 10.1021/jasms.0c00451. Epub 2021 Apr 5.
The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.
细胞周期是一个高度调控和进化保守的过程，其结果是细胞内容物的复制和复制的染色体均等分配到一对子细胞中。组蛋白是真核细胞染色质的基本结构成分，其翻译后修饰（PTMs）作为 DNA 读出和染色体浓缩的基准。与组蛋白 PTMs 失调相关的细胞周期异常是癌症等关键疾病的原因。监测组蛋白 PTMs 的变化可能为理解与细胞增殖的表观遗传调控相关的分子机制铺平道路。此前，我们实验室建立了一种使用多孔石墨碳（PGC）作为固定相来分析组蛋白 PTMs 的新型中-下分析工作流程，该方法与经典蛋白质组学一样，利用相同的反相色谱梯度分离，并与在线质谱（MS）相结合。在这里，我们将这种新型工作流程应用于细胞周期中 H3.1 和 H3.2 的组蛋白修饰的高通量分析。总的来说，我们鉴定了 1133 个独特修饰的经典组蛋白 H3 N 端尾部。与先前的发现一致，在有丝分裂（M）期，组蛋白 H3 的磷酸化显著增加。观察到组蛋白 H3 变体特异性和细胞周期依赖性 PTMs 的表达，这强调了将 H3.1 和 H3.2 组合在一起作为 H3 的必要性。我们证实了先前已知的 H3 PTM 串扰（例如，K9me-S10ph），并在此领域揭示了新的信息。这些发现意味着组合 PTMs 在细胞周期控制中发挥作用，它们可能作为增殖的标志物。
