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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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Characterization of the TgRSC8 locus and product.A. Northern hybridization indicates TgRSC8 is affected in C9. Total RNA from PruΔHPT (WT) or mutant C9 was probed with fragments of TgRSC8 and cat. TUB1 signal was used to assess equivalence of loading. B. Map of the insertion site in strain C9. The TgRSC8 locus is shown with exons (black), introns (white), and 5′ and 3′ UTRs (green and yellow, respectively) to scale. Insertion of 2 copies of pT230-TUB5/CAT (separated by vertical hatch marks) occurred in the 5′ UTR of TgRSC8. The cat gene and UTRs (red) are indicated on the plasmid cartoon, including the direction of transcription from the TUB1 promoter (arrow). C. Transcripts from luciferase reporter constructs. Maps of possible full-length transcripts from constructs containing a monocistronic LUC gene (LUC), polycistronic LUC encoded downstream of cat (cat-LUC), and polycistronic LUC with a partial TgRSC8 5′ UTR (green) between cat and LUC (cat-TgRSC8-LUC) are shown to scale. Additional noncoding sequences, including the TUB1 5′ and 3′ UTR (red and orange, respectively), and SAG1 3′ UTR (blue) are indicated. Vector sequences are shown as black lines. D. Luciferase reporters indicate translation occurs at the downstream locus of a polycistronic transcript in T. gondii. Activity is shown for reporter construct transformants whose transcripts are diagrammed in C, and is displayed as photons per second per tachyzoite. Shown is the average and standard deviations of three independent experiments.
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pone-0019570-g002: Characterization of the TgRSC8 locus and product.A. Northern hybridization indicates TgRSC8 is affected in C9. Total RNA from PruΔHPT (WT) or mutant C9 was probed with fragments of TgRSC8 and cat. TUB1 signal was used to assess equivalence of loading. B. Map of the insertion site in strain C9. The TgRSC8 locus is shown with exons (black), introns (white), and 5′ and 3′ UTRs (green and yellow, respectively) to scale. Insertion of 2 copies of pT230-TUB5/CAT (separated by vertical hatch marks) occurred in the 5′ UTR of TgRSC8. The cat gene and UTRs (red) are indicated on the plasmid cartoon, including the direction of transcription from the TUB1 promoter (arrow). C. Transcripts from luciferase reporter constructs. Maps of possible full-length transcripts from constructs containing a monocistronic LUC gene (LUC), polycistronic LUC encoded downstream of cat (cat-LUC), and polycistronic LUC with a partial TgRSC8 5′ UTR (green) between cat and LUC (cat-TgRSC8-LUC) are shown to scale. Additional noncoding sequences, including the TUB1 5′ and 3′ UTR (red and orange, respectively), and SAG1 3′ UTR (blue) are indicated. Vector sequences are shown as black lines. D. Luciferase reporters indicate translation occurs at the downstream locus of a polycistronic transcript in T. gondii. Activity is shown for reporter construct transformants whose transcripts are diagrammed in C, and is displayed as photons per second per tachyzoite. Shown is the average and standard deviations of three independent experiments.

Mentions: Insertion of disrupting plasmid pT/230-TUB/5CAT in C9 occurred upstream of the predicted translational start site of TgRSC8. Southern hybridization results show that the plasmid is present in two copies as a head-to-tail tandem repeat in a single insertion site (data not shown). To determine if insertion occurred within the transcribed region of TgRSC8, northern hybridization was performed on total RNA from PruΔHPT and C9 tachyzoites. When probed with a fragment of predicted exon 1 of TgRSC8 (Fig. S1), a transcript of 4.5 kb was detected in the wild-type strain, while C9 showed a transcript both larger in size and in relative abundance, as determined by probing for the T. gondii housekeeping gene encoding α-tubulin (TUB1, TgME49_116400) as a loading control (Fig. 2A). Sequencing and rapid amplification of cDNA ends (RACE) performed on the wild-type TgRSC8 transcript indicated a coding region of 2343 bp, matching the annotation for TgME49_086920, and a 5′ untranslated region of 715 bp. Two products were identified by 3′ RACE, indicating 3′ UTR sizes of 216 bp or 853 bp, the latter of which matches the predicted 3′ UTR on ToxoDB and more closely aligns with the total transcript size detected by hybridization. Plasmid insertion in C9 occurred within the 5′ UTR, 135 bp downstream of the transcriptional start site. Plasmid sequences rescued from C9 indicated that the T. gondii selectable marker of pT/230-TUB/5CAT was juxtaposed upstream of the TgRSC8 open reading frame (ORF). This bacterial-derived 663 bp chloramphenicol acetyltransferase (cat) gene is transcribed from a constitutive T. gondii TUB1 promoter, and employs the TUB1 5′ UTR, and SAG1 3′ UTR and downstream sequence. When probed with the E. coli cat coding sequences, no transcript is detected in PruΔHPT, however, two transcripts are detected in the C9 transformant (Fig. 2A). The cat transcript at 1.3 kb corresponds with cat expressed from the inserted plasmid, and the second transcript at 5.8 kb matches the size of the transcript detected with the TgRSC8 probe. These data indicate that in strain C9, transcription of TgRSC8 originated within the inserted plasmid, creating a fused cat-TgRSC8 transcript (Fig. 2B). The cat-TgRSC8 transcript is upregulated relative to the TgRSC8 transcript alone, likely due to transcription from the constitutive TUB1 promoter.


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

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

Characterization of the TgRSC8 locus and product.A. Northern hybridization indicates TgRSC8 is affected in C9. Total RNA from PruΔHPT (WT) or mutant C9 was probed with fragments of TgRSC8 and cat. TUB1 signal was used to assess equivalence of loading. B. Map of the insertion site in strain C9. The TgRSC8 locus is shown with exons (black), introns (white), and 5′ and 3′ UTRs (green and yellow, respectively) to scale. Insertion of 2 copies of pT230-TUB5/CAT (separated by vertical hatch marks) occurred in the 5′ UTR of TgRSC8. The cat gene and UTRs (red) are indicated on the plasmid cartoon, including the direction of transcription from the TUB1 promoter (arrow). C. Transcripts from luciferase reporter constructs. Maps of possible full-length transcripts from constructs containing a monocistronic LUC gene (LUC), polycistronic LUC encoded downstream of cat (cat-LUC), and polycistronic LUC with a partial TgRSC8 5′ UTR (green) between cat and LUC (cat-TgRSC8-LUC) are shown to scale. Additional noncoding sequences, including the TUB1 5′ and 3′ UTR (red and orange, respectively), and SAG1 3′ UTR (blue) are indicated. Vector sequences are shown as black lines. D. Luciferase reporters indicate translation occurs at the downstream locus of a polycistronic transcript in T. gondii. Activity is shown for reporter construct transformants whose transcripts are diagrammed in C, and is displayed as photons per second per tachyzoite. Shown is the average and standard deviations of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
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pone-0019570-g002: Characterization of the TgRSC8 locus and product.A. Northern hybridization indicates TgRSC8 is affected in C9. Total RNA from PruΔHPT (WT) or mutant C9 was probed with fragments of TgRSC8 and cat. TUB1 signal was used to assess equivalence of loading. B. Map of the insertion site in strain C9. The TgRSC8 locus is shown with exons (black), introns (white), and 5′ and 3′ UTRs (green and yellow, respectively) to scale. Insertion of 2 copies of pT230-TUB5/CAT (separated by vertical hatch marks) occurred in the 5′ UTR of TgRSC8. The cat gene and UTRs (red) are indicated on the plasmid cartoon, including the direction of transcription from the TUB1 promoter (arrow). C. Transcripts from luciferase reporter constructs. Maps of possible full-length transcripts from constructs containing a monocistronic LUC gene (LUC), polycistronic LUC encoded downstream of cat (cat-LUC), and polycistronic LUC with a partial TgRSC8 5′ UTR (green) between cat and LUC (cat-TgRSC8-LUC) are shown to scale. Additional noncoding sequences, including the TUB1 5′ and 3′ UTR (red and orange, respectively), and SAG1 3′ UTR (blue) are indicated. Vector sequences are shown as black lines. D. Luciferase reporters indicate translation occurs at the downstream locus of a polycistronic transcript in T. gondii. Activity is shown for reporter construct transformants whose transcripts are diagrammed in C, and is displayed as photons per second per tachyzoite. Shown is the average and standard deviations of three independent experiments.
Mentions: Insertion of disrupting plasmid pT/230-TUB/5CAT in C9 occurred upstream of the predicted translational start site of TgRSC8. Southern hybridization results show that the plasmid is present in two copies as a head-to-tail tandem repeat in a single insertion site (data not shown). To determine if insertion occurred within the transcribed region of TgRSC8, northern hybridization was performed on total RNA from PruΔHPT and C9 tachyzoites. When probed with a fragment of predicted exon 1 of TgRSC8 (Fig. S1), a transcript of 4.5 kb was detected in the wild-type strain, while C9 showed a transcript both larger in size and in relative abundance, as determined by probing for the T. gondii housekeeping gene encoding α-tubulin (TUB1, TgME49_116400) as a loading control (Fig. 2A). Sequencing and rapid amplification of cDNA ends (RACE) performed on the wild-type TgRSC8 transcript indicated a coding region of 2343 bp, matching the annotation for TgME49_086920, and a 5′ untranslated region of 715 bp. Two products were identified by 3′ RACE, indicating 3′ UTR sizes of 216 bp or 853 bp, the latter of which matches the predicted 3′ UTR on ToxoDB and more closely aligns with the total transcript size detected by hybridization. Plasmid insertion in C9 occurred within the 5′ UTR, 135 bp downstream of the transcriptional start site. Plasmid sequences rescued from C9 indicated that the T. gondii selectable marker of pT/230-TUB/5CAT was juxtaposed upstream of the TgRSC8 open reading frame (ORF). This bacterial-derived 663 bp chloramphenicol acetyltransferase (cat) gene is transcribed from a constitutive T. gondii TUB1 promoter, and employs the TUB1 5′ UTR, and SAG1 3′ UTR and downstream sequence. When probed with the E. coli cat coding sequences, no transcript is detected in PruΔHPT, however, two transcripts are detected in the C9 transformant (Fig. 2A). The cat transcript at 1.3 kb corresponds with cat expressed from the inserted plasmid, and the second transcript at 5.8 kb matches the size of the transcript detected with the TgRSC8 probe. These data indicate that in strain C9, transcription of TgRSC8 originated within the inserted plasmid, creating a fused cat-TgRSC8 transcript (Fig. 2B). The cat-TgRSC8 transcript is upregulated relative to the TgRSC8 transcript alone, likely due to transcription from the constitutive TUB1 promoter.

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