The long-term goal of this research is to investigate transcription regulatory mechanisms in Apicomplexan protozoa, a phylum of intracellular parasites that cause significant medical and economic burden. Toxoplasma gondii causes congenital birth defects and is an AIDS opportunist. Virtually nothing is known about how gene expression is regulated in the Apicomplexa, but the observation that that the antiprotozoal apicidin targets the histone modification machinery argues that investigating these mechanisms may reveal novel therapeutic opportunities. Since changes in gene expression coincide with critical changes during parasite development, elucidating the network of transcriptional regulators will also reveal a great deal about parasite biology. We have cloned a novel homologue of GCN5 in T. gondii (TgGCN5), the catalytic component of an acetylase complex that increases transcription by covalently modifying histones. Our hypothesis is: TgGCN5 regulates gene expression in T. gondii and its unusual N-terminal domain is critical to this function.
Specific aims are: 1) Discover genes regulated by TgGCN5 and characterize GCN5-dependent promoters. To accomplish this, microarrays and proteomics are being used to compare GCN5 expression mutants to wildtype parasites. DNA-binding proteins that associate with TgGCN5-dependent promoters will be identified in order to understand how the acetylase complex is recruited. 2) Define role(s) for the unusual N-terminal extension of TgGCN5. This region of TgGCN5 is responsible for nuclear localization in T. gondii and the precise sequence is currently being mapped since it will be novel. The Nterminal domain will also be examined for roles in modulating enzymatic activity and/or forming the multisubunit GCN5 acetylase complex. 3) Identify proteins constituting the GCN5 acetylase complex. Coimmunoprecipitation, yeast two-hybrid screens, and bioinformatics will be employed to achieve this aim. Several components of a GCN5 acetylase complex in yeast are essential for viability, underscoring the significance of investigating these analogues in T. gondii. Collectively, these aims will answer how TgGCN5 and its assocated molecules regulate transcription, significantly contributing to our knowledge about the gene regulatory circuitry in this group of important pathogens. ? ? ?
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