A Dynamic and Combinatorial Histone Code Drives Malaria Parasite Asexual and Sexual Development

Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these “histone codes” are the result of cross talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 77 PTMs on H3 and H3.3, of which 34 are newly identified in P. falciparum. Coexisting PTMs with unique stage distinctions were identified, indicating that many of these combinatorial PTMs are associated with specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2, which we tied to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with coexisting partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher-order gene regulation for distinct proliferation and differentiation processes.