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Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation.

Rooney PJ, Neal LM, Knoll LJ - PLoS ONE (2011)

Bottom Line: In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts.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.Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

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.

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TgRSC8 expression correlates with BAG1 expression.TgRSC8 and BAG1 levels were determined for C9 (blue), a vector control strain (VC2, green) and C9 complement compN (red) parasites subjected to bradyzoite induction conditions, by flow cytometry. TgRSC8 levels are displayed on the x-axis and BAG1 levels on the y-axis, in arbitrary units. Three independent experiments were performed, showing similar results. Results of a representative experiment are shown.
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pone-0019570-g006: TgRSC8 expression correlates with BAG1 expression.TgRSC8 and BAG1 levels were determined for C9 (blue), a vector control strain (VC2, green) and C9 complement compN (red) parasites subjected to bradyzoite induction conditions, by flow cytometry. TgRSC8 levels are displayed on the x-axis and BAG1 levels on the y-axis, in arbitrary units. Three independent experiments were performed, showing similar results. Results of a representative experiment are shown.

Mentions: In the C9 mutant, TgRSC8 is the downstream gene on a polycistronic transcript, and thus it may show reduced translational efficiency. Additionally, complementation of the C9 mutant with TgRSC8 restored BAG1 expression to wild-type levels. These data suggested a reduction in expression of TgRSC8 in C9 as compared to the wild type. To facilitate quantification of TgRSC8, we used the 3AD6 monoclonal antibody. A reduction in TgRSC8 in C9 was detected by immunofluorescence using monoclonal anti-TgRSC8 antibody (Fig. 5A). To quantify the reduction in TgRSC8 protein in the C9 mutant, vector control strains containing disrupting plasmid pT/230-TUB/5CAT were compared to C9 and complemented mutants by western immunoblot and flow cytometry, examining populations of both tachyzoites and in vitro bradyzoites. TgRSC8 was reduced in populations of both stages of C9 parasites relative to either vector controls or complements (Fig. 5B,C; data not shown). Western blots, flow cytometry, and immunofluorescence microscopy revealed that this reduction was minimal though detectable in C9 tachyzoites, whereas the reduction of TgRSC8 in C9 bradyzoites was more pronounced (Fig. 5B; data not shown). Complementation with the TgRSC8 locus increased expression of this protein in both tachyzoites and bradyzoites over that of the mutant C9 (Fig. 5A,B,C; data not shown). The level of TgRSC8 in bradyzoites correlated with expression of BAG1 in strain C9. Parasites showing a loss of BAG1 expression similarly demonstrated a loss of TgRSC8, though some BAG1 positive C9 parasites were also found to have reduced TgRSC8 expression, a variability not seen in BAG1 positive parasites of the vector control strain (Fig. 5A). Flow cytometric analyses of C9, a C9 complement and vector control bradyzoites using 3AD6 ascites showed that increased TgRSC8 expression was associated with increased BAG1 expression for all strains tested (Fig. 6). Taken together, these data demonstrate that despite increased levels of transcript, insertion within the 5′UTR of TgRSC8 resulted in a reduction in translated protein, and that TgRSC8 facilitates wild-type expression of BAG1 from in vitro bradyzoites.


Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation.

Rooney PJ, Neal LM, Knoll LJ - PLoS ONE (2011)

TgRSC8 expression correlates with BAG1 expression.TgRSC8 and BAG1 levels were determined for C9 (blue), a vector control strain (VC2, green) and C9 complement compN (red) parasites subjected to bradyzoite induction conditions, by flow cytometry. TgRSC8 levels are displayed on the x-axis and BAG1 levels on the y-axis, in arbitrary units. Three independent experiments were performed, showing similar results. Results of a representative experiment are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104990&req=5

pone-0019570-g006: TgRSC8 expression correlates with BAG1 expression.TgRSC8 and BAG1 levels were determined for C9 (blue), a vector control strain (VC2, green) and C9 complement compN (red) parasites subjected to bradyzoite induction conditions, by flow cytometry. TgRSC8 levels are displayed on the x-axis and BAG1 levels on the y-axis, in arbitrary units. Three independent experiments were performed, showing similar results. Results of a representative experiment are shown.
Mentions: In the C9 mutant, TgRSC8 is the downstream gene on a polycistronic transcript, and thus it may show reduced translational efficiency. Additionally, complementation of the C9 mutant with TgRSC8 restored BAG1 expression to wild-type levels. These data suggested a reduction in expression of TgRSC8 in C9 as compared to the wild type. To facilitate quantification of TgRSC8, we used the 3AD6 monoclonal antibody. A reduction in TgRSC8 in C9 was detected by immunofluorescence using monoclonal anti-TgRSC8 antibody (Fig. 5A). To quantify the reduction in TgRSC8 protein in the C9 mutant, vector control strains containing disrupting plasmid pT/230-TUB/5CAT were compared to C9 and complemented mutants by western immunoblot and flow cytometry, examining populations of both tachyzoites and in vitro bradyzoites. TgRSC8 was reduced in populations of both stages of C9 parasites relative to either vector controls or complements (Fig. 5B,C; data not shown). Western blots, flow cytometry, and immunofluorescence microscopy revealed that this reduction was minimal though detectable in C9 tachyzoites, whereas the reduction of TgRSC8 in C9 bradyzoites was more pronounced (Fig. 5B; data not shown). Complementation with the TgRSC8 locus increased expression of this protein in both tachyzoites and bradyzoites over that of the mutant C9 (Fig. 5A,B,C; data not shown). The level of TgRSC8 in bradyzoites correlated with expression of BAG1 in strain C9. Parasites showing a loss of BAG1 expression similarly demonstrated a loss of TgRSC8, though some BAG1 positive C9 parasites were also found to have reduced TgRSC8 expression, a variability not seen in BAG1 positive parasites of the vector control strain (Fig. 5A). Flow cytometric analyses of C9, a C9 complement and vector control bradyzoites using 3AD6 ascites showed that increased TgRSC8 expression was associated with increased BAG1 expression for all strains tested (Fig. 6). Taken together, these data demonstrate that despite increased levels of transcript, insertion within the 5′UTR of TgRSC8 resulted in a reduction in translated protein, and that TgRSC8 facilitates wild-type expression of BAG1 from in vitro bradyzoites.

Bottom Line: In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts.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.Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

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.

Show MeSH
Related in: MedlinePlus