The Histone Code of Toxoplasma gondii Comprises Conserved and Unique Posttranslational Modifications

S.C.N. and F.-Y.C. contributed equally to this article. ABSTRACT Epigenetic gene regulation has emerged as a major mechanism for gene regulation in all eukaryotes. Histones are small, basic proteins that constitute the major protein component of chromatin, and posttranslational modifications (PTM) of histones are essential for epigenetic gene regulation. The different combinations of histone PTM form the histone code for an organism, marking functional units of chromatin that recruit macromolecular complexes that govern chromatin structure and regulate gene expression. To characterize the repertoire of Toxoplasma gondii histone PTM, we enriched histones using stan-dard acid extraction protocols and analyzed themwith several complementary middle-down and bottom-up proteomic ap-proaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy colli-sional dissociation (HCD), and/or electron transfer dissociation (ETD) fragmentation. We identified 249 peptides with unique combinations of PTM that comprise the T. gondii histone code. T. gondii histones share a high degree of sequence conservation with human histones, and many modifications are conserved between these species. In addition, T. gondii histones have uniqu