Browsing by Subject "Histone"

Now showing 1 - 4 of 4
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    Characterization of protein arginine methyltransferase of TgPRMT5 in Toxoplasma gondii
    (Springer Nature, 2019-05-08) Liu, Min; Li, Fen-Xiang; Li, Chun-Yuan; Li, Xiao-Cong; Chen, Long-Fei; Wu, Kun; Yang, Pei-Liang; Lai, Zhi-Fa; Liu, Ting-kai; Sullivan, William J., Jr.; Cui, Liwang; Chen, Xiao-Guang; Pharmacology and Toxicology, School of Medicine
    BACKGROUND: Protein arginine methylation is a prevalent post-translational modification. The protein arginine methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. METHODS: We tagged the 3' end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has arginine methyltransferase activity. RESULTS: IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylarginines and symmetric dimethylarginines. CONCLUSIONS: We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 protein may be involved in tachyzoite-bradyzoite transformation.
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    In vivo pulse-chase in C. elegans reveals intestinal histone turnover changes upon starvation
    (bioRxiv, 2025-02-16) Borchers, Christopher; Osburn, Kara; Roh, Hyun Cheol; Aoki, Scott T.; Biochemistry and Molecular Biology, School of Medicine
    The ability to study protein dynamics and function in the authentic context of a multicellular organism is paramount to better understand biological phenomena in animal health and disease. Pulse-chase of self-labeling fusion protein tags provide the opportunity to label proteins of interest and track those proteins over time. There are currently several challenges associated with performing in vivo protein pulse-chase in animals, such as cost, reproducibility, and accurate detection methods. The C. elegans model organism has attributes that alleviate many of these challenges. This work tests the feasibility of applying the Halo modified enzyme (HaloTag) for in vivo protein pulse-chase in C. elegans. HaloTag intestinal histone reporters were created in the worm and used to demonstrate that reporter protein could be efficiently pulse-labeled by soaking animals in ligand. Labeled protein stability could be monitored over time by fluorescent confocal microscopy. Further investigation revealed reporter protein stability was dependent on the animal's nutritional state. Chromatin Immunoprecipitation and sequencing (ChIP-seq) of the reporters showed incorporation in chromatin with little change hours into starvation, implying a lack of chromatin regulation at the time point tested. Collectively, this work presents a straightforward method to label and track proteins of interest in C. elegans that can address a multitude of biological questions surrounding protein stability and dynamics in this animal model.
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    Toxoplasma H2A Variants Reveal Novel Insights into Nucleosome Composition and Functions for this Histone Family
    (Elsevier, 2009) Dalmasso, Maria C.; Onyango, David O.; Naguleswaran, Arunasalam; Sullivan, William J., Jr.; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here we report the characterization of three H2A histones, a canonical H2A1 and variants H2AX and H2AZ. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)φ (φ denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We also show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We also found that h2ax expression, but not h2a1 and h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are actively expressed and h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this life cycle stage. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.
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    Toxoplasma histone acetylation remodelers as novel drug targets
    (Taylor & Francis, 2012) Vanagas, Laura; Jeffers, Victoria; Bogado, Silvina S.; Dalmasso, Maria C.; Sullivan, William J., Jr.; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    Toxoplasma gondii is a leading cause of neurological birth defects and a serious opportunistic pathogen. The authors and others have found that Toxoplasma uses a unique nucleosome composition supporting a fine gene regulation together with other factors. Post-translational modifications in histones facilitate the establishment of a global chromatin environment and orchestrate DNA-related biological processes. Histone acetylation is one of the most prominent post-translational modifications influencing gene expression. Histone acetyltransferases and histone deacetylases have been intensively studied as potential drug targets. In particular, histone deacetylase inhibitors have activity against apicomplexan parasites, underscoring their potential as a new class of antiparasitic compounds. In this review, we summarize what is known about Toxoplasma histone acetyltransferases and histone deacetylases, and discuss the inhibitors studied to date. Finally, the authors discuss the distinct possibility that the unique nucleosome composition of Toxoplasma, which harbors a nonconserved H2Bv variant histone, might be targeted in novel therapeutics directed against this parasite.