Diversifying the histone code: defining the role of non-canonical phosphorylation in chromatin structure

已知组蛋白的翻译后修饰（PTM）指导染色质结构并因此指导基因转录。这种所谓的“组蛋白密码”假设不同组蛋白上的PTM的组合是调节DNA包装的驱动力，从而调节不同基因可用于转录或“沉默”的能力。利用我们实验室最近开发的技术，使用质谱法在细胞范围内鉴定组氨酸、赖氨酸、精氨酸等非典型磷酸化位点（NCP），我们鉴定了组蛋白（如H1.4、H2 B、H3）和自身调节组蛋白PTM状态的酶（如HDAC 1/2/7、CBX 1、KAT 6A）上的NCP位点。这些NCP位点的鉴定提出了这样一种可能性，即我们所理解的组蛋白密码很可能是不完整的。目前用于理解组蛋白修饰的方法依赖于基于酸的组蛋白提取，这与这些类型的不稳定NCP事件不相容。或者，使用PTM位点特异性抗体，由于紧密定位的PTM引起的表位不相容性，或相同位点上的不同PTM，可能使富集偏向并妨碍鉴定。该项目旨在重新定义组蛋白密码及其调控，使用与保留组蛋白及其调控酶上酸不稳定磷酸化位点相容的方法。我们还将探讨这种重新定义的组蛋白密码和染色质结构之间的关系。

Post-translational modifications (PTMs) of histones are known to direct chromatin structure and thus gene transcription. This so called "histone code" hypothesises that the combination of PTMs on different histones are a driving force that regulate DNA packing and thus the ability of different genes to be accessible for transcription or 'silenced'. Using technology recently developed in our lab for the cell-wide identification of sites of non-canonical phosphorylation (NCP) on e.g. histidine, lysine, arginine using mass spectrometry, we have identified NCP sites on histone proteins (e.g. H1.4, H2B, H3), and on enzymes that themselves regulate histone PTM status (e.g. HDAC1/2/7, CBX1, KAT6A). Identification of these NCP sites raises the possibility that the histone code, as we understand it, may very well be incomplete. The current methods for understanding histone modifications rely on acid-based histone extraction that is incompatible with these types of labile NCP events. Alternatively, PTM site specific antibodies are employed, that due either to epitope incompatibility arising from closely localised PTMs, or different PTMs on the same site, may bias enrichment and preclude identification. This project aims to re-define the histone code and its regulation, using methods that are compatible with retaining sites of acid-labile phosphorylation on histones and their regulatory enzymes. We will also explore the relationship between this re-defined histone code and chromatin structure.