New Approaches and Insights into the Gene Expression of Toxoplasma gondii
Croken, Matthew McKnight
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Toxoplasma gondii is an intracellular parasite. Each strain of the parasite has phenotypic differences relating to virulence, host preference, and competence for life cycle progression. Using high-throughput RNA sequencing, we assayed the transcriptomes of three different strains of T. gondii, RH (type I), PLK (type II), and CTG (type III), each during two developmental stages, tachyzoite and bradyzoite. By examining these parasites' mRNA levels, we can understand how differential gene expression contributes to observed differences in phenotypes as well as those genes involved in bradyzote differentiation for each strain of the parasite.;Each stage of the parasite's complex life cycle has a distinct pattern of steady-state mRNA levels. Here, we demonstrate that different strains of Toxoplasma possess vastly different transcriptomes. These differences are greater than changes in gene expression associated with life cycle progression. We identify cell cycle regulated genes as key markers of differentiation.;We find several hundred genes whose expression differs either between strains. Most of these genes are differentially expressed between strains regardless of life cycle stage, suggesting that inter-strain differences relate to more than virulence or differentiation competence.;GSEA is a pathway analysis tool which uses pre-defined sets of genes with related functions. To develop gene sets for T. gondii, we leveraged existing expression data to group together genes which are co-regulated during development, the cell cycle, co-localized to the same organelle, or members of the same metabolic pathway.;Using GSEA and our newly developed gene sets, we identify biological pathways which differ between sample conditions. Our gene sets provide an objective method to quantify important parasite phenomena as opposed to relying on the presence or absence of a handful of life cycle "marker" proteins.;Finally, we develop methodologies to study epigenetic factors that modify chromatin and coordinate expression. Using chromatin immunoprecipitation, we defined the genome-wide position of chromatin-associated proteins and examined the co-localization of modified nucleosomes, histone-modifying enzymes, and annotated genes. The result is allows us to create a framework of interacting histone marks which regulate chromatin structure and transcription.