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

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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.