GCN5-mediated transcription in AIDS pathogen Toxoplasma

Indiana University Indianapolis, Indianapolis IN

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Abstract

ABSTRACT Epigenetics and histone modifications play key roles in the pathogenic processes of protozoal parasites. We have made several important observations regarding histone acetylation in the AIDS opportunist, Toxoplasma gondii. Using chromatin immunoprecipitation (ChIP), we found that histone acetylation correlates with gene expression pertinent to parasite differentiation, the process that underlies pathogenesis. While characterizing the GCN5-family histone acetyltransferase (HAT), we discovered that Toxoplasma harbors a second GCN5 HAT (named TgGCN5-A and -B, respectively). While both TgGCN5s mediate the histone acetylation that we have linked to parasite differentiation, these HATs possess unique N-terminal extensions, implying they have independent roles in regulating gene expression. We have established that the N-terminal extensions are critical for nuclear localization and protein-protein interactions. Furthermore, our yeast two-hybrid screen with the N-terminal extension of TgGCN5-A has revealed unique parasite-specific interacting proteins that have domains typical of transcription regulators. To begin addressing the cellular roles of each TgGCN5, we have attempted to generate gene knockouts. We have established that TgGCN5-A is not required for tachyzoite replication, but may be involved in differentiation or reactivation of latent infection. Our preliminary data also suggests that TgGCN5-B may be essential for parasite viability. In short, GCN5-mediated histone acetylation is not only relevant to understanding parasite differentiation, but also holds promise as a drug target. We hypothesize that the two TgGCN5 HATs play key independent roles in Toxoplasma pathogenesis by forming distinct complexes with novel proteins that regulate gene expression. Our specific aims include (1) Determine the roles of TgGCN5 HATs in pathogenesis; (2) Elucidate differences in TgGCN5 complexes by identifying proteins that bind to the unique N-terminal extensions; (3) Define the mechanism by which each TgGCN5 is recruited to target gene promoters. The proposed research expands our previous work and capitalizes on the novel reagents, techniques, and GCN5 transgenic parasites that we have developed. The results will have a positive impact on the field by defining mechanisms of GCN5-mediated gene regulation and by revealing components of the parasite[unreadable]s transcriptional regulatory circuitry that have significant value as potential drug targets. Thus, our results will fundamentally advance the fields of parasitology and eukaryotic gene expression.