Mol. Cells 2014; 37(5): 389-398
Published online May 14, 2014
https://doi.org/10.14348/molcells.2014.0032
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: mrhee@cnu.ac.kr (MR); rohyunju@cnu.ac.kr (HR)
Siah2 is a zebrafish homologue of mammalian Siah family. Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between
Keywords antivin/Lft1, body patterning, FoxH1/Fast1, Nodal, Siah2, zebrafish
Siah is the mammalian homologue of the protein encoded by
Vertebrate body patterning is initiated during gastrulation as endoderm, mesoderm and ectoderm are induced along the dorsoventral (DV) and anteroposterior (AP) axes. Fgf, Wnt and Nodal signals initiate early dorsoventral pattern in zebrafish embryos (Schier and Talbot, 2005). After fertilization the dorsal determinants are translocated to the dorsal side through the subcortical microtubules. The dorsal determinants lead to the activation of maternal Wnt signaling. Maternal Wnt, especially Wnt8a in the case of zebrafish activates β-catenin, a transcriptional activator, to regulate expression of several genes important for the early embryonic body patterning (Huelsken and Birchmeier, 2001; Lu et al., 2011). Gore et al. (2005) proposed that Squint (Sqt/Ndr1), one of the Nodal-related proteins, acts as a potent dorsal determinant through the differential localization as early as four cells stage. Previous works showed that Siah1 functions as a negative regulator of the Wnt signaling by down-regulating β-catenin stability (Liu et al., 2001; Matsuzawa and Reed, 2001). Siah2 up-regulates Fgf downstream signaling by destructing Sprouty (a Fgf-dependent ERK inhibitor) in a phosphorylation-independent manner while Siah1 does not (Nadeau et al., 2006; Qi et al., 2008). It thus appears that Siah family plays critical roles in vertebrate body axis formation by modulating various signaling pathways.
Several genetic studies and overexpression experiments indicated that
We have previously isolated and characterized a zebrafish Siah and designated it Siaz (renamed to Siah2l following ZFIN designation. But we will call the Siah2l as Siah2 in this report to avoid confusion) (Ro et al., 2003; 2005). In this report, we demonstrated that Siah as a critical intracellular Nodal modulator acts on upstream of Fast1/FoxH1 transcriptional factor for the maintenance of Nodal homeostasis. We revealed that the functional RING domain of Siah2 is exclusively required for antagonizing the function of Nodal inhibitors. Positive roles of Siah for Nodal signaling were reinforced by gene depletion experiments. Knocking-down of Siah1 and Siah2 with specific morpholinos (MO) induced the morphants partially mimicking the morphology of Nodal defective mutants. Collectively, these results clarify the roles of Siah family in Nodal signaling.
Fish and embryos were maintained essentially as described in the zebrafish book (Westerfield, 1995).
293T cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented 10% fetal bovine serum (FBS). Cells in culture dishes were transfected with various plasmid constructs (pFlag/Siah2, pFlag/Siha2 Mu, pcGlobin2/Siah2, pcGlobin2/Fast-1, pcGlobin2/Fast1-SID) using FuGene6 transfection reagent (Roche). After 24 h, cells were harvested and then the cell pellet was used for further assay.
We co-transfected 3TP-Luc reporter construct (0.5 μg) with indicated DNAs into 293T cells using calcium phosphate precipitation technique. Total amount of transfected DNA was equalized with control vectors without inserts. Transfected 293 cells were treated with TGF-β (1 ng/ml) for 24 h, and lysed for luciferase assay using the Dual Luciferase system (Promega).
Antisense riboprobes were constructed using appropriate RNA polymerase following the instructions (Ambion).
For the microinjection, cDNA constructs were subcloned into the pcGlobin2 vector (Ro et al., 2004b). mRNAs for injection were synthesized from the vector constructs linearized with the appropriate restriction enzymes, using the mMESSAGE mMACHINE T7 kit (Ambion Inc.) according to the manufacturers instruction. After purification as following the manufacturers recommendation, mRNAs were dissolved in diethylpyrocarbo-nate (DEPC)-treated 0.1 M KCl. Before injection, the mRNAs were diluted to various concentrations and 1 μl of mRNA was used to inject approximately 400 embryos. The mRNA and MOs were pressure injected into the yolk of 1?2 cell stage of embryos and the injected embryos were raised in 1/3 Ringers solution (39 mM NaCl, 0.97 mM KCl, 1.8 mM CaCl2, 1.7 mM Hepes, pH 7.2). Injections were performed three to four times to pool the data. To reduce the p53-dependent off-targeting effects of
Total RNA was isolated from zebrafish embryos using easy-BLUE™ (INTRON, Inc.) and 1 μg of RNA was used for RT-PCR.
For the amino acids substitution of Siah2 H107A and C110A, mega-primer was generated by PCR using
Interestingly,
To ask if the augmented
We confirmed the antagonizing effects of Siah2 on Atv/Lft2 by analyzing expression patterns of
To investigate whether the Siah2 reconstituted
Because Nodal transduction pathway is mediated by intracellular Smad and FoxH1 (also called Fast1) protein complex (Attisano et al., 2001), we also examined whether Siah2 requires the intracellular elements for its biological functions. We initially analyzed the effects of WT and dominant negative form of Fast1 on
These studies suggest that Siah2 requires Fast1 transcription factor to stimulate transcription of Nodal downstream genes, such as
Siah proteins bind E2 ubiquitin conjugating enzymes
To explore the
Highly conserved Siah homologues have been identified in fly, mouse, zebrafish and human. Here we reported that Siah augmented a sufficient quantity of
One of the plausible targets of Siah in Nodal signaling is TIEG1, a transcriptional repressor of
Another putative target of Siah in Nodal signaling is Zic2. Houston and Wylie (2005) showed that maternally supplied
Iratni et al. (2002) reported that transcriptional corepressor DRAP1 interacts with Fast-1 and inhibits DNA binding. Interestingly, the DRAP1-deficient mice showed expanded
We previously identified a novel Siah2 interacting partner designated as Sinup using yeast two-hybrid screening with Siah2 as bait (Ro et al., 2005). Overexpression of
A previous report showed that T84 epithelial cells treated with TGF-β down regulates Siah1 and Siah2 expression level when the cells were cultured three-dimensionally (Juuti-Uusitalo et al., 2006). Recently, Liao et al. (2012) also reported that undifferentiated small intestinal crypt cells (IEC-6 cell line) in response to TGF-β stimuli increased the expression of miRNA-146b, which induced
Mol. Cells 2014; 37(5): 389-398
Published online May 31, 2014 https://doi.org/10.14348/molcells.2014.0032
Copyright © The Korean Society for Molecular and Cellular Biology.
Nami Kang1,3, Minho Won2,3, Myungchull Rhee1,*, and Hyunju Ro1,*
1Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea, 2Program in Genomics of Differentiation, Eunice Kennedy Shiver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA, 3These authors contributed equally to this work.
Correspondence to:*Correspondence: mrhee@cnu.ac.kr (MR); rohyunju@cnu.ac.kr (HR)
Siah2 is a zebrafish homologue of mammalian Siah family. Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between
Keywords: antivin/Lft1, body patterning, FoxH1/Fast1, Nodal, Siah2, zebrafish
Siah is the mammalian homologue of the protein encoded by
Vertebrate body patterning is initiated during gastrulation as endoderm, mesoderm and ectoderm are induced along the dorsoventral (DV) and anteroposterior (AP) axes. Fgf, Wnt and Nodal signals initiate early dorsoventral pattern in zebrafish embryos (Schier and Talbot, 2005). After fertilization the dorsal determinants are translocated to the dorsal side through the subcortical microtubules. The dorsal determinants lead to the activation of maternal Wnt signaling. Maternal Wnt, especially Wnt8a in the case of zebrafish activates β-catenin, a transcriptional activator, to regulate expression of several genes important for the early embryonic body patterning (Huelsken and Birchmeier, 2001; Lu et al., 2011). Gore et al. (2005) proposed that Squint (Sqt/Ndr1), one of the Nodal-related proteins, acts as a potent dorsal determinant through the differential localization as early as four cells stage. Previous works showed that Siah1 functions as a negative regulator of the Wnt signaling by down-regulating β-catenin stability (Liu et al., 2001; Matsuzawa and Reed, 2001). Siah2 up-regulates Fgf downstream signaling by destructing Sprouty (a Fgf-dependent ERK inhibitor) in a phosphorylation-independent manner while Siah1 does not (Nadeau et al., 2006; Qi et al., 2008). It thus appears that Siah family plays critical roles in vertebrate body axis formation by modulating various signaling pathways.
Several genetic studies and overexpression experiments indicated that
We have previously isolated and characterized a zebrafish Siah and designated it Siaz (renamed to Siah2l following ZFIN designation. But we will call the Siah2l as Siah2 in this report to avoid confusion) (Ro et al., 2003; 2005). In this report, we demonstrated that Siah as a critical intracellular Nodal modulator acts on upstream of Fast1/FoxH1 transcriptional factor for the maintenance of Nodal homeostasis. We revealed that the functional RING domain of Siah2 is exclusively required for antagonizing the function of Nodal inhibitors. Positive roles of Siah for Nodal signaling were reinforced by gene depletion experiments. Knocking-down of Siah1 and Siah2 with specific morpholinos (MO) induced the morphants partially mimicking the morphology of Nodal defective mutants. Collectively, these results clarify the roles of Siah family in Nodal signaling.
Fish and embryos were maintained essentially as described in the zebrafish book (Westerfield, 1995).
293T cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented 10% fetal bovine serum (FBS). Cells in culture dishes were transfected with various plasmid constructs (pFlag/Siah2, pFlag/Siha2 Mu, pcGlobin2/Siah2, pcGlobin2/Fast-1, pcGlobin2/Fast1-SID) using FuGene6 transfection reagent (Roche). After 24 h, cells were harvested and then the cell pellet was used for further assay.
We co-transfected 3TP-Luc reporter construct (0.5 μg) with indicated DNAs into 293T cells using calcium phosphate precipitation technique. Total amount of transfected DNA was equalized with control vectors without inserts. Transfected 293 cells were treated with TGF-β (1 ng/ml) for 24 h, and lysed for luciferase assay using the Dual Luciferase system (Promega).
Antisense riboprobes were constructed using appropriate RNA polymerase following the instructions (Ambion).
For the microinjection, cDNA constructs were subcloned into the pcGlobin2 vector (Ro et al., 2004b). mRNAs for injection were synthesized from the vector constructs linearized with the appropriate restriction enzymes, using the mMESSAGE mMACHINE T7 kit (Ambion Inc.) according to the manufacturers instruction. After purification as following the manufacturers recommendation, mRNAs were dissolved in diethylpyrocarbo-nate (DEPC)-treated 0.1 M KCl. Before injection, the mRNAs were diluted to various concentrations and 1 μl of mRNA was used to inject approximately 400 embryos. The mRNA and MOs were pressure injected into the yolk of 1?2 cell stage of embryos and the injected embryos were raised in 1/3 Ringers solution (39 mM NaCl, 0.97 mM KCl, 1.8 mM CaCl2, 1.7 mM Hepes, pH 7.2). Injections were performed three to four times to pool the data. To reduce the p53-dependent off-targeting effects of
Total RNA was isolated from zebrafish embryos using easy-BLUE™ (INTRON, Inc.) and 1 μg of RNA was used for RT-PCR.
For the amino acids substitution of Siah2 H107A and C110A, mega-primer was generated by PCR using
Interestingly,
To ask if the augmented
We confirmed the antagonizing effects of Siah2 on Atv/Lft2 by analyzing expression patterns of
To investigate whether the Siah2 reconstituted
Because Nodal transduction pathway is mediated by intracellular Smad and FoxH1 (also called Fast1) protein complex (Attisano et al., 2001), we also examined whether Siah2 requires the intracellular elements for its biological functions. We initially analyzed the effects of WT and dominant negative form of Fast1 on
These studies suggest that Siah2 requires Fast1 transcription factor to stimulate transcription of Nodal downstream genes, such as
Siah proteins bind E2 ubiquitin conjugating enzymes
To explore the
Highly conserved Siah homologues have been identified in fly, mouse, zebrafish and human. Here we reported that Siah augmented a sufficient quantity of
One of the plausible targets of Siah in Nodal signaling is TIEG1, a transcriptional repressor of
Another putative target of Siah in Nodal signaling is Zic2. Houston and Wylie (2005) showed that maternally supplied
Iratni et al. (2002) reported that transcriptional corepressor DRAP1 interacts with Fast-1 and inhibits DNA binding. Interestingly, the DRAP1-deficient mice showed expanded
We previously identified a novel Siah2 interacting partner designated as Sinup using yeast two-hybrid screening with Siah2 as bait (Ro et al., 2005). Overexpression of
A previous report showed that T84 epithelial cells treated with TGF-β down regulates Siah1 and Siah2 expression level when the cells were cultured three-dimensionally (Juuti-Uusitalo et al., 2006). Recently, Liao et al. (2012) also reported that undifferentiated small intestinal crypt cells (IEC-6 cell line) in response to TGF-β stimuli increased the expression of miRNA-146b, which induced
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