title: Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation.
abstract: The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Peggy J
authors.initials: PJ
authors.lastname: Rooney
authors.forename: Lori M
authors.initials: LM
authors.lastname: Neal
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Knoll
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D042002:Chromatin Assembly and Disassembly / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000235:genetics / Q000378:metabolism*
chemical_list: C106179:BAG1 protein, Toxoplasma; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D015800:Protozoan Proteins