Identification and Characterization of a Mef2 Transcriptional Activator in Schistosome Parasites

Milligan, John N.; Jolly, Emmitt R.

doi:10.1371/journal.pntd.0001443

Cited by 5 publications

(16 citation statements)

References 74 publications

(104 reference statements)

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“…We previously identified and characterized Mef2 in schistosomes (SmMef2) [40], a conserved transcriptional activator that is essential for myogenesis in Drosophila [41]. Mef2 also has diverse functions regulating cellular differentiation, morphogenesis and proliferation [61], [62].…”

Section: Resultsmentioning

confidence: 99%

“…SmMef2 levels are highest in 4-hour schistosomula relative sporocysts, cercariae, and adult worms [40]. We cloned the SmMef2 gene under control of the CMV promoter, as was previously described for the truncation mutant, SmMef2,133 , and overexpressed SmMef2.. To distinguish between expression of SmMef2 and the mutant SmMef2,133 , we designed DNA oligonucleotides that recognize SmMef2 (Figure 1D, oligonucleotides f and g) but that do not recognize SmMef2,133 transcript.…”

Section: Resultsmentioning

confidence: 99%

“…Subcloning was performed using the In-Fusion HD Cloning kit (Clontech, Mountainview, CA). The full transcript of the mCherry gene from the transposon vector pKM225 (GenBank: HQ386859.1), the first 399 bp (133 amino acids) of SmMef2 , and the wild-type SmMef2 [40] (NCBI accession number: JN900476) were amplified by PCR using Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA) with three sets of primers: oEJ1020 forward (5′-TCA CGC GTG GTA CCT CTA GAA TGG TGA GCA AGG GCG AGG AG) and oEJ1021 reverse (5′-GCC CGG GTC GAC TCT AGA TTA CTT GTA CAG CTC GTC CAT GCC), oEJ1026 forward (5′-CAC TAT AGG CTA GCC TCG AGA TGG GTC GCA AAA AAA TAC TCA TC) and oEJ1027 reverse (5′-GCG TGA ATT CTC GAG CTA CGG TGT TTT AGT TCC TGT TCG TAT), and oLS197b forward (5′-ATA GGC TAG CCT CGA GAT GGG TCG CAA AAA AAT ACT CAT CA-3′) and oLS198 reverse (5′-TAA AGG GAA GCG GCC GCT CAA AGG TGG CGC ACA CGT TTA AGA-3′), respectively. mCherry and truncated Sm Mef2 (Sm Mef2,133 ) amplicons were subcloned into the pCI-neo plasmid (Promega, Madison, WI) at the XbaI and XhoI sites, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The melt-curve analysis of each pair of primers showed that only one specific product was amplified by each reaction. The relative gene expression was calculated by the ΔΔC t method according to the formula: expression rate = 2 −ΔΔCt and cyclophilin was used as an endogenous control gene [40]. Experimental data were verified by Student's t-test, and a p-value less than 0.05 was considered to be statistically significant [50].…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we characterized the schistosome myocyte enhancer factor (SmMef2) [40], a DNA-binding transcriptional activator that is important for cellular development, morphogenesis and survival in mammals and Drosophila (for review on Mef2, see [41]). We identified potential SmMef2 DNA binding elements in the promoters of wingless-type MMTV integration site family members 1 and 2 ( Wnt1 and Wnt2 ) homologs.…”

Section: Introductionmentioning

confidence: 99%

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Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2

2013

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Schistosomiasis is a serious global problem and the second most devastating parasitic disease following malaria. Parasitic worms of the genus Schistosoma are the causative agents of schistosomiasis and infect more than 240 million people worldwide. The paucity of molecular tools to manipulate schistosome gene expression has made an understanding of genetic pathways in these parasites difficult, increasing the challenge of identifying new potential drugs for treatment. Here, we describe the use of a formulation of polyethyleneimine (PEI) as an alternative to electroporation for the efficacious transfection of genetic material into schistosome parasites. We show efficient expression of genes from a heterologous CMV promoter and from the schistosome Sm23 promoter. Using the schistosome myocyte enhancer factor 2 (SmMef2), a transcriptional activator critical for myogenesis and other developmental pathways, we describe the development of a dominant-negative form of the schistosome Mef2. Using this mutant, we provide evidence that SmMef2 may regulate genes in the WNT pathway. We also show that SmMef2 regulates its own expression levels. These data demonstrate the use of PEI to facilitate effective transfection of nucleic acids into schistosomes, aiding in the study of schistosome gene expression and regulation, and development of genetic tools for the characterization of molecular pathways in these parasites.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2

2013

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Function of Nanos1 gene in the development of reproductive organs of Schistosoma japonicum

et al. 2017

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Nanos is a necessary factor in the differentiation and migration of primordial germ cells. It is closely associated with the development of genitalia in a wide range of species. We questioned whether Nanos was involved in the reproductive organ development of Schistosoma japonicum. Firstly, by in situ hybridization, S. japonicum Nanos1 (SjNanos1) gene was expressed mainly in reproductive organs of S. japonicum. Then, the paired schistosome of 28 days post-infection (dpi) was transfected with SjNanos1 small interfering RNA three times and cultured in vitro for 10 days. SjNanos1 expression suppression in the mRNA and protein levels were confirmed compared to that of the controls. The morphological changes in reproductive organs and egg production were observed after SjNanos1 gene knockdown. The results observed by confocal laser scanning microscopy showed significant changes in the morphology of reproductive organs of parasites, especially the female ovaries, vitellarium, and the male testes, after RNAi. In addition, SjNanos1 silencing also induced the reduction of eggs, and affected the changes of reproduction-related genes, like Pumilio, CNOT6L, and Fs800. Therefore, our findings demonstrate that the SjNanos1 gene is essential in the development of reproductive organs and the egg production of S. japonicum.

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Tyrosine kinase 4 is involved in the reproduction of the platyhelminth parasite Schistosoma japonicum

et al. 2017

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BackgroundSchistosomiasis is one of the most common parasitic diseases affecting millions of humans and animals worldwide. Understanding the signal transduction pathways and the molecular basis of reproductive regulation in schistosomes is critically important for developing new strategies for preventing and treating these infections. Syk kinases regulate the proliferation, differentiation, morphogenesis, and survival of various types of cells and have been identified in invertebrates. Tyrosine kinase 4 (TK4), a member of the Syk kinase family, plays a pivotal role in gametogenesis in S. mansoni, affecting the development of the testis and ovaries in this parasite. The role of TK4, however, in the reproduction of S. japonicum is poorly understood. MethodsHere, the complete coding sequence of TK4 gene in S. japonicum (SjTK4) was cloned and characterized. The expression of SjTK4 was analyzed at different life-cycle stages and in various tissues of S. japonicum by qPCR. Piceatannol, a Syk kinase inhibitor, was applied to S. japonicum in vitro. The piceatannol-induced morphological changes of the parasites were observed using confocal laser scanning microscopy and the alterations in important egg-shell synthesis-related genes were examined using qPCR analyses.Results SjTK4 mRNA was differentially expressed throughout the life-cycle of S. japonicum. SjTK4 mRNA was highly expressed in the ovary and testis of S. japonicum, with the level of gene expression significantly higher in males than in females. The expression levels of some important egg-shell synthesis related genes were higher in the piceatannol-treated groups than in the vehicle-treated control group and the number of eggs and germ cells also decreased in a concentration-dependent manner. Importantly, large pore-like structures can be found in the testis and ovaries of males and females after treating with piceatannol.ConclusionThe results suggest that SjTK4 may play an important role in regulating gametogenesis of S. japonicum. The findings may help better understand the fundamental biology of S. japonicum. Moreover, the effect of S. japonicum treatment by piceatannol provides us with a new idea that inhibition of SjTK4 signaling pathway can effectively retard the development of the testis and ovaries.

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Identification and Characterization of a Mef2 Transcriptional Activator in Schistosome Parasites

Cited by 5 publications

References 74 publications

Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2

Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2

Function of Nanos1 gene in the development of reproductive organs of Schistosoma japonicum

Tyrosine kinase 4 is involved in the reproduction of the platyhelminth parasite Schistosoma japonicum

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