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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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Anti-TgRSC8 antibodies detect a 84kDa nuclear protein.A. Anti-TgRSC8 monoclonal antibodies 3AD6 and 1DE10 detect an 84 kDa protein in extracts of either tachyzoites (T) or bradyzoites (B) of the vector control stain VC2 by western immunoblot. Markers of molecular mass are shown at the right in kDa. Blots were reprobed to indicate reactivity of β-tubulin (TUB2) and BAG1 as controls for loading and bradyzoite transition, respectively. B. TgRSC8 is a nuclear protein in both tachyzoites and bradyzoites. TgRSC8 was detected in wild-type parasites using 1DE10 antibody (green). DNA is detected by DAPI (blue). Tachyzoites are indicated by detection of the surface marker SAG1, while bradyzoites are shown by Dolichos biflorus agglutinin (DbA) reactivity (red). At left are DIC images, including a 5 µm scale indicator (black bar), and merged panels are shown on the right.
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pone-0019570-g004: Anti-TgRSC8 antibodies detect a 84kDa nuclear protein.A. Anti-TgRSC8 monoclonal antibodies 3AD6 and 1DE10 detect an 84 kDa protein in extracts of either tachyzoites (T) or bradyzoites (B) of the vector control stain VC2 by western immunoblot. Markers of molecular mass are shown at the right in kDa. Blots were reprobed to indicate reactivity of β-tubulin (TUB2) and BAG1 as controls for loading and bradyzoite transition, respectively. B. TgRSC8 is a nuclear protein in both tachyzoites and bradyzoites. TgRSC8 was detected in wild-type parasites using 1DE10 antibody (green). DNA is detected by DAPI (blue). Tachyzoites are indicated by detection of the surface marker SAG1, while bradyzoites are shown by Dolichos biflorus agglutinin (DbA) reactivity (red). At left are DIC images, including a 5 µm scale indicator (black bar), and merged panels are shown on the right.

Mentions: To further characterize TgRSC8, monoclonal antibodies were generated against the purified recombinant protein. By denaturing western immunoblot, antibodies 1DE10 and 3AD6 reacted strongly with an 84 kDa protein in both tachyzoite and in vitro developed bradyzoite protein extracts (Fig. 4A). This corresponds to the protein mass based on translation of the predicted ORF. These anti-TgRSC8 monoclonal antibodies were used to examined the subcellular localization of TgRSC8. In S. cerevisiae, Rsc8p localizes to the nucleus, and directly complexes to DNA and histones to control gene expression. Using either monoclonal antibody for detection within T. gondii tachyzoites and in vitro developed bradyzoites, TgRSC8 appeared in the same compartment with nuclear DNA as identified by both DAPI and reactivity with antiserum against the major T. gondii H2B histone, H2Bv (Fig. 4B; data not shown) [33]. Although TgRSC8 was clearly nuclear, it did not co-localize precisely with either DAPI-detected nuclear DNA, or H2Bv. To confirm the specificity of the monoclonal antibodies and localization of TgRSC8, the complemented C9 transformant expressing amino-terminal HA tagged TgRSC8 was assessed using anti-HA antibody-based detection (Fig. 3). As this HA tagged version of TgRSC8 complemented the C9 mutant, this suggests correct localization of the protein. Amino-terminal HA tagged TgRSC8 also localized to the parasite nucleus in a pattern similar to that seen using the monoclonal antibodies against TgRSC8 (Fig. S2). While addition of the HA tag to the carboxy-terminus of TgRSC8 rendered it non-functional with regard to the effects on BAG1 expression (Fig. 3B), it also localized to the nucleus in both tachyzoites and in vitro bradyzoites (data not shown). These results demonstrate the specificity of the monoclonal antibodies for detection of TgRSC8, which appear within the nucleus of T. gondii.


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

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

Anti-TgRSC8 antibodies detect a 84kDa nuclear protein.A. Anti-TgRSC8 monoclonal antibodies 3AD6 and 1DE10 detect an 84 kDa protein in extracts of either tachyzoites (T) or bradyzoites (B) of the vector control stain VC2 by western immunoblot. Markers of molecular mass are shown at the right in kDa. Blots were reprobed to indicate reactivity of β-tubulin (TUB2) and BAG1 as controls for loading and bradyzoite transition, respectively. B. TgRSC8 is a nuclear protein in both tachyzoites and bradyzoites. TgRSC8 was detected in wild-type parasites using 1DE10 antibody (green). DNA is detected by DAPI (blue). Tachyzoites are indicated by detection of the surface marker SAG1, while bradyzoites are shown by Dolichos biflorus agglutinin (DbA) reactivity (red). At left are DIC images, including a 5 µm scale indicator (black bar), and merged panels are shown on the right.
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Related In: Results  -  Collection

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pone-0019570-g004: Anti-TgRSC8 antibodies detect a 84kDa nuclear protein.A. Anti-TgRSC8 monoclonal antibodies 3AD6 and 1DE10 detect an 84 kDa protein in extracts of either tachyzoites (T) or bradyzoites (B) of the vector control stain VC2 by western immunoblot. Markers of molecular mass are shown at the right in kDa. Blots were reprobed to indicate reactivity of β-tubulin (TUB2) and BAG1 as controls for loading and bradyzoite transition, respectively. B. TgRSC8 is a nuclear protein in both tachyzoites and bradyzoites. TgRSC8 was detected in wild-type parasites using 1DE10 antibody (green). DNA is detected by DAPI (blue). Tachyzoites are indicated by detection of the surface marker SAG1, while bradyzoites are shown by Dolichos biflorus agglutinin (DbA) reactivity (red). At left are DIC images, including a 5 µm scale indicator (black bar), and merged panels are shown on the right.
Mentions: To further characterize TgRSC8, monoclonal antibodies were generated against the purified recombinant protein. By denaturing western immunoblot, antibodies 1DE10 and 3AD6 reacted strongly with an 84 kDa protein in both tachyzoite and in vitro developed bradyzoite protein extracts (Fig. 4A). This corresponds to the protein mass based on translation of the predicted ORF. These anti-TgRSC8 monoclonal antibodies were used to examined the subcellular localization of TgRSC8. In S. cerevisiae, Rsc8p localizes to the nucleus, and directly complexes to DNA and histones to control gene expression. Using either monoclonal antibody for detection within T. gondii tachyzoites and in vitro developed bradyzoites, TgRSC8 appeared in the same compartment with nuclear DNA as identified by both DAPI and reactivity with antiserum against the major T. gondii H2B histone, H2Bv (Fig. 4B; data not shown) [33]. Although TgRSC8 was clearly nuclear, it did not co-localize precisely with either DAPI-detected nuclear DNA, or H2Bv. To confirm the specificity of the monoclonal antibodies and localization of TgRSC8, the complemented C9 transformant expressing amino-terminal HA tagged TgRSC8 was assessed using anti-HA antibody-based detection (Fig. 3). As this HA tagged version of TgRSC8 complemented the C9 mutant, this suggests correct localization of the protein. Amino-terminal HA tagged TgRSC8 also localized to the parasite nucleus in a pattern similar to that seen using the monoclonal antibodies against TgRSC8 (Fig. S2). While addition of the HA tag to the carboxy-terminus of TgRSC8 rendered it non-functional with regard to the effects on BAG1 expression (Fig. 3B), it also localized to the nucleus in both tachyzoites and in vitro bradyzoites (data not shown). These results demonstrate the specificity of the monoclonal antibodies for detection of TgRSC8, which appear within the nucleus of T. gondii.

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