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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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SWIRM and SANT domains of TgRSC8.Alignment of SWIRM and SANT domains of TgRSC8 with yeast Rsc8p (ScRsc8p) and Swi3p (ScSwi3p; Genbank accession numbers NP116695 and NP012359, respectively). At left of the sequence is the first amino acid position number of the regions shown. Asterisks below the alignments indicate sites of amino acid identity, while dots indicate conservative differences. Amino acids in boxes were modified in TgRSC8 in this study.
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pone-0019570-g001: SWIRM and SANT domains of TgRSC8.Alignment of SWIRM and SANT domains of TgRSC8 with yeast Rsc8p (ScRsc8p) and Swi3p (ScSwi3p; Genbank accession numbers NP116695 and NP012359, respectively). At left of the sequence is the first amino acid position number of the regions shown. Asterisks below the alignments indicate sites of amino acid identity, while dots indicate conservative differences. Amino acids in boxes were modified in TgRSC8 in this study.

Mentions: Screening of a T. gondii insertional mutant library identified strains defective in tachyzoite to bradyzoite differentiation in vitro [27]. Identification of the disrupted locus in one of these mutants, strain C9, was performed by plasmid rescue. Sequences flanking the plasmid insertion site were compared to ToxoDB and showed identity to locus TgME49_086920. Alignments of the predicted 84 kDa protein using ClustalW showed the TgME49_086920 protein to have 29.4% similarity and 16.5% identity to yeast Rsc8p, and 35.0% similarity and 17.7% identity to yeast Swi3p. Rsc8p and Swi3p both contain SWIRM and SANT domains. The alpha-helical SWIRM domain is predicted to mediate interactions with both protein and DNA in the assembly of chromatin-protein complexes. This domain within Swi3p was shown to directly bind free DNA as well as nucleosomal DNA. Amino acids D374 and N392 were shown to be critical for this process [28]. SWIRM was identified within TgME49_086920 from amino acids 266 to 356 (PFAM-A expect value of 1.6×10-20, Pfam v24.0 http://pfam.sanger.ac.uk [29]), sharing key residues with its yeast homologs, including the aforementioned aspartic acid and asparagine (Fig. 1). The SANT domain of Rsc8p was shown to associate with histones, with amino acid W546 critical to the structure [30]. The amino acid R564 in the SANT domain of Swi3p provided a positive charge necessary for function [31]. The SANT domain of TgME49_086920 was located between amino acids 491 and 537 (PFAM-A expect value of 6.8×10−10) and showed conservation of the yeast W546 and R564 residues (Fig. 1). As TgME49_086920 was the only Swi3p/Rsc8p homolog identified in the T. gondii genome, we named this locus TgRSC8.


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

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

SWIRM and SANT domains of TgRSC8.Alignment of SWIRM and SANT domains of TgRSC8 with yeast Rsc8p (ScRsc8p) and Swi3p (ScSwi3p; Genbank accession numbers NP116695 and NP012359, respectively). At left of the sequence is the first amino acid position number of the regions shown. Asterisks below the alignments indicate sites of amino acid identity, while dots indicate conservative differences. Amino acids in boxes were modified in TgRSC8 in this study.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019570-g001: SWIRM and SANT domains of TgRSC8.Alignment of SWIRM and SANT domains of TgRSC8 with yeast Rsc8p (ScRsc8p) and Swi3p (ScSwi3p; Genbank accession numbers NP116695 and NP012359, respectively). At left of the sequence is the first amino acid position number of the regions shown. Asterisks below the alignments indicate sites of amino acid identity, while dots indicate conservative differences. Amino acids in boxes were modified in TgRSC8 in this study.
Mentions: Screening of a T. gondii insertional mutant library identified strains defective in tachyzoite to bradyzoite differentiation in vitro [27]. Identification of the disrupted locus in one of these mutants, strain C9, was performed by plasmid rescue. Sequences flanking the plasmid insertion site were compared to ToxoDB and showed identity to locus TgME49_086920. Alignments of the predicted 84 kDa protein using ClustalW showed the TgME49_086920 protein to have 29.4% similarity and 16.5% identity to yeast Rsc8p, and 35.0% similarity and 17.7% identity to yeast Swi3p. Rsc8p and Swi3p both contain SWIRM and SANT domains. The alpha-helical SWIRM domain is predicted to mediate interactions with both protein and DNA in the assembly of chromatin-protein complexes. This domain within Swi3p was shown to directly bind free DNA as well as nucleosomal DNA. Amino acids D374 and N392 were shown to be critical for this process [28]. SWIRM was identified within TgME49_086920 from amino acids 266 to 356 (PFAM-A expect value of 1.6×10-20, Pfam v24.0 http://pfam.sanger.ac.uk [29]), sharing key residues with its yeast homologs, including the aforementioned aspartic acid and asparagine (Fig. 1). The SANT domain of Rsc8p was shown to associate with histones, with amino acid W546 critical to the structure [30]. The amino acid R564 in the SANT domain of Swi3p provided a positive charge necessary for function [31]. The SANT domain of TgME49_086920 was located between amino acids 491 and 537 (PFAM-A expect value of 6.8×10−10) and showed conservation of the yeast W546 and R564 residues (Fig. 1). As TgME49_086920 was the only Swi3p/Rsc8p homolog identified in the T. gondii genome, we named this locus TgRSC8.

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