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Browsing by Author "Weiss, Louis M."
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Item ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst(National Academy of Sciences, 2013) Radke, Joshua B.; Lucas, Olivier; De Silva, Erandi K.; Ma, YanFen; Sullivan, William J., Jr.; Weiss, Louis M.; Llinas, Manuel; White, Michael W.; Pharmacology and Toxicology, School of MedicineCellular differentiation leading to formation of the bradyzoite tissue cyst stage is the underlying cause of chronic toxoplasmosis. Consequently, mechanisms responsible for controlling development in the Toxoplasma intermediate life cycle have long been sought. Here, we identified 15 Toxoplasma mRNAs induced in early bradyzoite development that encode proteins with apicomplexan AP2 (ApiAP2) DNA binding domains. Of these 15 mRNAs, the AP2IX-9 mRNA demonstrated the largest expression increase during alkaline-induced differentiation. At the protein level, we found that AP2IX-9 was restricted to the early bradyzoite nucleus and is repressed in tachyzoites and in mature bradyzoites from 30-d infected animals. Conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation and conferred alkaline pH-resistant growth, whereas disruption of the AP2IX-9 gene increased tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs, including the canonical bradyzoite marker, bradyzoite antigen 1 (BAG1). Using protein binding microarrays, we established the AP2 domain of AP2IX-9 binds a CAGTGT DNA sequence motif and is capable of binding cis-regulatory elements controlling the BAG1 and bradyzoite-specific nucleoside triphosphatase (B-NTPase) promoters. The effect of AP2IX-9 on BAG1 expression was direct because this factor inhibits expression of a firefly luciferase reporter under the control of the BAG1 promoter in vivo, and epitope-tagged AP2IX-9 can be immunoprecipitated with the BAG1 promoter in parasite chromatin. Altogether, these results indicate AP2IX-9 restricts Toxoplasma commitment to develop the mature bradyzoite tissue cyst.Item The Histone Code of Toxoplasma gondii Comprises Conserved and Unique Posttranslational Modifications(American Society for Microbiology, 2013-12-10) Nardelli, Sheila C.; Che, Fa-Yun; Silmon de Monerri, Natalie C.; Xiao, Hui; Nieves, Edward; Madrid-Aliste, Carlos; Angel, Sergio O.; Sullivan, William J., Jr.; Angeletti, Ruth H.; Kim, Kami; Weiss, Louis M.; Pharmacology and Toxicology, School of MedicineEpigenetic 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 standard acid extraction protocols and analyzed them with several complementary middle-down and bottom-up proteomic approaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy collisional 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 unique modifications not previously identified in other species. Finally, T. gondii histones are modified by succinylation, propionylation, and formylation, recently described histone PTM that have not previously been identified in parasitic protozoa. The characterization of the T. gondii histone code will facilitate in-depth analysis of how epigenetic regulation affects gene expression in pathogenic apicomplexan parasites and identify a new model system for elucidating the biological functions of novel histone PTM. Importance: Toxoplasma gondii is among the most common parasitic infections in humans. The transition between the different stages of the T. gondii life cycle are essential for parasite virulence and survival. These differentiation events are accompanied by significant changes in gene expression, and the control mechanisms for these transitions have not been elucidated. Important mechanisms that are involved in the control of gene expression are the epigenetic modifications that have been identified in several eukaryotes. T. gondii has a full complement of histone-modifying enzymes, histones, and variants. In this paper, we identify over a hundred PTM and a full repertoire of PTM combinations for T. gondii histones, providing the first large-scale characterization of the T. gondii histone code and an essential initial step for understanding how epigenetic modifications affect gene expression and other processes in this organism.