Department of Chemistry, University of California, Riverside, California 92521-0403, USA.
Biochemistry. 2010 Jun 29;49(25):5236-43. doi: 10.1021/bi1001322.
Core histones are susceptible to a variety of post-translational modifications (PTMs), among which methylation and acetylation play critical roles in various chromatin-dependent processes. The nature and biological functions of these PTMs have been extensively studied in plants, animals, and yeasts. In contrast, the histone modifications in Neurospora crassa, a convenient model organism for multicellular eukaryotes, remained largely undefined. In this study, we used several mass spectrometric techniques, coupled with HPLC separation and multiple-protease digestion, to identify the methylation and acetylation sites in core histones isolated from Neurospora. Electron transfer dissociation (ETD) was employed to fragment the heavily modified long N-terminal peptides. In addition, accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from trimethylation. Many modification sites conserved in other organisms were identified in Neurospora. In addition, some unique modification sites in histone H2B, including N-terminal alpha methylation, methylation at K3, and acetylation at K19, K28, and K29, were observed. Our analysis provides a potentially comprehensive picture of methylation and acetylation of core histones in Neurospora, which should serve as a foundation for future studies of the function of histone PTMs in this model organism.
核心组蛋白容易发生多种翻译后修饰（PTMs），其中甲基化和乙酰化在各种依赖染色质的过程中起着关键作用。这些 PTMs 的性质和生物学功能在植物、动物和酵母中得到了广泛研究。相比之下，秀丽隐杆线虫（Neurospora crassa）中的组蛋白修饰在很大程度上仍未得到定义，秀丽隐杆线虫是多细胞真核生物的一种方便的模式生物。在这项研究中，我们使用了几种质谱技术，结合 HPLC 分离和多种蛋白酶消化，来鉴定从秀丽隐杆线虫中分离出的核心组蛋白中的甲基化和乙酰化位点。电子转移解离（ETD）用于使修饰较多的长 N 端肽片段化。此外，片段离子的精确质量测量允许明确地区分乙酰化和三甲基化。在秀丽隐杆线虫中鉴定到了许多在其他生物中保守的修饰位点。此外，还观察到组蛋白 H2B 中的一些独特修饰位点，包括 N 端α甲基化、K3 位甲基化以及 K19、K28 和 K29 位乙酰化。我们的分析为秀丽隐杆线虫核心组蛋白的甲基化和乙酰化提供了一个潜在的全面图景，这应该为该模式生物中组蛋白 PTM 功能的未来研究奠定基础。
