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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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Effects of TgRSC8 reduction on transcript levels of bradyzoite-induced and constitutive genes.Q-PCR was performed on cDNA derived from in vitro bradyzoites of vector control strains VC1 (white bars) and VC2 (gray bars), TgRSC8 mutant C9 (black bars), and three complemented strains (comp1-3, patterned bars) to analyze transcript levels of bradyzoite-induced genes BAG1, LDH2 (panel A), SUSA1, ENO1 (panel B), a bradyzoite-induced HSP70, BRP1, BGR1 and SAG2X (panel C). Transcripts of constitutive genes GRA2 and GAPDH were also quantified (panel D). Transcript levels were expressed as a percentage of the level of housekeeping gene TUB1 per sample. Shown are the averages and standard deviations of three experiments, performed on cDNAs derived from independent RNA samples.
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pone-0019570-g007: Effects of TgRSC8 reduction on transcript levels of bradyzoite-induced and constitutive genes.Q-PCR was performed on cDNA derived from in vitro bradyzoites of vector control strains VC1 (white bars) and VC2 (gray bars), TgRSC8 mutant C9 (black bars), and three complemented strains (comp1-3, patterned bars) to analyze transcript levels of bradyzoite-induced genes BAG1, LDH2 (panel A), SUSA1, ENO1 (panel B), a bradyzoite-induced HSP70, BRP1, BGR1 and SAG2X (panel C). Transcripts of constitutive genes GRA2 and GAPDH were also quantified (panel D). Transcript levels were expressed as a percentage of the level of housekeeping gene TUB1 per sample. Shown are the averages and standard deviations of three experiments, performed on cDNAs derived from independent RNA samples.

Mentions: S. cerevisiae Rsc8p is known to impact transcription of many genes in yeast. As BAG1 protein expression in C9 was reduced in bradyzoites, the steady state levels of several transcripts previously described as induced in bradyzoites, as well as two constitutively transcribed genes, were determined for strain C9, three complemented strains and two vector control strains by Q-PCR in bradyzoites. The genes analyzed included bradyzoite-induced genes encoding BAG1 (TgME49_059020), LDH2 (TgME49_091040), SUSA1 (TgME49_078080), ENO1 (TgME49_068860), SAG2X (SRS49B, TgME49_007140), BRP1 (TgME49_114250), and BGR1 (TgME49_053330), which represent a diverse group from metabolic enzymes to surface markers [34-38; Buchholz and Boothroyd, personal communication]. As BAG1 is a heat shock protein, we sought an additional heat shock protein target for study. Analysis indicating T. gondii expresses a bradyzoite-induced HSP70 was performed using methods now known to detect three closely related HSP70s (TgME49_073760, _111720, and _051780) [39]. Locus TgME49_111720 showed the largest number of bradyzoite expressed sequence tags, and was selected for analysis (ToxoDB). Transcript levels of housekeeping genes TUB1 and GAPDH, and the gene encoding dense granule protein 2 (GRA2, TgME49_027620), predicted to be expressed equivalently in both stages in wild-type T. gondii, were also assessed (Fig. S3A; ToxoDB) [40]. In keeping with microscopic evidence of BAG1 protein expression, the BAG1 transcript was decreased in the TgRSC8 mutant and restored to the levels of the control strains in complements (Fig. 7A). Steady-state transcript levels of the bradyzoite-induced genes LDH2, SUSA1 and ENO1 transcripts were also significantly reduced in the mutant relative to vector controls, and increased in complemented strains (Fig. 7A,B). No effect was seen on transcript levels of GAPDH, or GRA2, which were equivalently expressed in both tachyzoites and bradyzoites (Fig. 7D; data not shown).


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

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

Effects of TgRSC8 reduction on transcript levels of bradyzoite-induced and constitutive genes.Q-PCR was performed on cDNA derived from in vitro bradyzoites of vector control strains VC1 (white bars) and VC2 (gray bars), TgRSC8 mutant C9 (black bars), and three complemented strains (comp1-3, patterned bars) to analyze transcript levels of bradyzoite-induced genes BAG1, LDH2 (panel A), SUSA1, ENO1 (panel B), a bradyzoite-induced HSP70, BRP1, BGR1 and SAG2X (panel C). Transcripts of constitutive genes GRA2 and GAPDH were also quantified (panel D). Transcript levels were expressed as a percentage of the level of housekeeping gene TUB1 per sample. Shown are the averages and standard deviations of three experiments, performed on cDNAs derived from independent RNA samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019570-g007: Effects of TgRSC8 reduction on transcript levels of bradyzoite-induced and constitutive genes.Q-PCR was performed on cDNA derived from in vitro bradyzoites of vector control strains VC1 (white bars) and VC2 (gray bars), TgRSC8 mutant C9 (black bars), and three complemented strains (comp1-3, patterned bars) to analyze transcript levels of bradyzoite-induced genes BAG1, LDH2 (panel A), SUSA1, ENO1 (panel B), a bradyzoite-induced HSP70, BRP1, BGR1 and SAG2X (panel C). Transcripts of constitutive genes GRA2 and GAPDH were also quantified (panel D). Transcript levels were expressed as a percentage of the level of housekeeping gene TUB1 per sample. Shown are the averages and standard deviations of three experiments, performed on cDNAs derived from independent RNA samples.
Mentions: S. cerevisiae Rsc8p is known to impact transcription of many genes in yeast. As BAG1 protein expression in C9 was reduced in bradyzoites, the steady state levels of several transcripts previously described as induced in bradyzoites, as well as two constitutively transcribed genes, were determined for strain C9, three complemented strains and two vector control strains by Q-PCR in bradyzoites. The genes analyzed included bradyzoite-induced genes encoding BAG1 (TgME49_059020), LDH2 (TgME49_091040), SUSA1 (TgME49_078080), ENO1 (TgME49_068860), SAG2X (SRS49B, TgME49_007140), BRP1 (TgME49_114250), and BGR1 (TgME49_053330), which represent a diverse group from metabolic enzymes to surface markers [34-38; Buchholz and Boothroyd, personal communication]. As BAG1 is a heat shock protein, we sought an additional heat shock protein target for study. Analysis indicating T. gondii expresses a bradyzoite-induced HSP70 was performed using methods now known to detect three closely related HSP70s (TgME49_073760, _111720, and _051780) [39]. Locus TgME49_111720 showed the largest number of bradyzoite expressed sequence tags, and was selected for analysis (ToxoDB). Transcript levels of housekeeping genes TUB1 and GAPDH, and the gene encoding dense granule protein 2 (GRA2, TgME49_027620), predicted to be expressed equivalently in both stages in wild-type T. gondii, were also assessed (Fig. S3A; ToxoDB) [40]. In keeping with microscopic evidence of BAG1 protein expression, the BAG1 transcript was decreased in the TgRSC8 mutant and restored to the levels of the control strains in complements (Fig. 7A). Steady-state transcript levels of the bradyzoite-induced genes LDH2, SUSA1 and ENO1 transcripts were also significantly reduced in the mutant relative to vector controls, and increased in complemented strains (Fig. 7A,B). No effect was seen on transcript levels of GAPDH, or GRA2, which were equivalently expressed in both tachyzoites and bradyzoites (Fig. 7D; data not shown).

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