Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

Cho, Chao-Cheng; Su, Li‐Hsin; Huang, Ying‐Fong; Pan, Yu-Jiao; Sun, Chin-Hung

doi:10.1074/jbc.m111.298893

Cited by 17 publications

(24 citation statements)

References 88 publications

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“…Expression of variant surface proteins may be regulated by a microRNA mediated post transcriptional regulation system [29], but little is known of relative regulation in the CWP expression. Giardia encystation has been proposed to link to cell cycle regulation and Cdk2 pathway may be involved in activation of Myb2 and up-regulation of cwp genes [30], [31], [32].…”

Section: Introductionmentioning

confidence: 99%

DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

Lin

Weng

et al. 2013

PLoS Negl Trop Dis

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The protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.

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Section: Introductionmentioning

confidence: 99%

DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

Lin

Weng

et al. 2013

PLoS Negl Trop Dis

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“…This phenomenon suggests that TaCRK2 is likely to coordinate the parasite's [17]. The protozoan G. lamblia CDK2 localized in the cytoplasm participate in the cytoplasm participates in the increase in cyst formation and shows increased expression during encystation [8]. The members of P. falciparum CDK-like kinase (CLK) family, PfCLk-1 and PfCLK-2, are primarily localized in the nucleus, and the further dispersion of PfCLK-2 into the cytoplasm, indicates that PfCLKs participate in gene regulation via the post-transcriptional modi cation of mRNA in the malaria blood stage [36].…”

Section: Discussionmentioning

confidence: 95%

“…During schizogamy, the E. tenella proliferates within host cells at a high division rate [4], this process is similar to cell cycles in other eukaryotes regulated by cyclin-dependent kinase (CDKs) [5]. CDKs are a kind of serine/threonine kinases that regulate the activity of substrate proteins through phosphorylation [6,7], which regulates cell cycle progression, proliferation, and differentiation and modulate transcription [8][9][10][11]. CDK2 plays a pivotal role in regulating G1/S and S/G2 transitions during the cell cycle [12,13].…”

Section: Introductionmentioning

confidence: 99%

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Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

Zhou

Ding

Yang

et al. 2020

Preprint

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Background：Coccidiosis caused by Eimeria tenella infection, directly or indirectly leads to great loss to poultry industry. With the emergence of drug-resistance in chicken coccidia, it is imperative to develop new drugs. Cyclin-dependent kinases (CDKs) regulate cell cycle progression in numerous organisms by acting as key molecular switches. Results: In the present study, a diclazuril anticoccidiosis animal model was established and CDK-related kinase 2 (CRK2) in the second-generation merozoite of E. tenella (EtCRK2) gene was amplified through reverse transcription-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli Rosetta (DE3). Purified recombinant protein was used for antiserum preparation. Subsequently, EtCRK2 transcription and translation levels were detected through quantitative real-time PCR and Western blot analysis, respectively. The localization of EtCRK2 in merozoites was examined via immunofluorescence techniques. Results showed that the mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localized in the cytoplasm of merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. Conclusions: This study demonstrated that the anticoccidial drug diclazuril against E. tenella by affecting the expression pattern of EtCRK2 molecule, and EtCRK2 may be used as a candidate target for new drug development.

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“…Far Western blot analysis, a non-antibody method, was further used to confirm this interaction between the MYB2-N and TOP3β-HA to avoid the non-specific problem (figure 7e). The MYB2-N can be phosphorylated by CDK2, which is involved in inducing encystation [6]. TOP3β can bind to AT-rich elements of both the constitutive ran gene and encystation-induced cwp genes (figure 6).…”

Section: Discussionmentioning

confidence: 99%

DNA topoisomerase IIIβ promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia

Sun

Weng

et al. 2020

Open Biol.

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Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3β) with similarity to the mammalian topoisomerase IIIβ. We found that TOP3β was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3β can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3β can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3β increased expression of cwp1 - 3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3β. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3β gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3β may be functionally conserved, and involved in inducing Giardia cyst formation.

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Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

Cited by 17 publications

References 88 publications

DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia

Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

DNA topoisomerase IIIβ promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia

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