Volume 46, Issue 5, Pages 650-661 (June 2012) RNF12 Controls Embryonic Stem Cell Fate and Morphogenesis in Zebrafish Embryos by Targeting Smad7 for Degradation  Long Zhang, Huizhe Huang, FangFang Zhou, Joost Schimmel, Cristina Gontan Pardo, Tingting Zhang, Tahsin Stefan Barakat, Kelly-Ann Sheppard, Craig Mickanin, Jeff A. Porter, Alfred C.O. Vertegaal, Hans van Dam, Joost Gribnau, Chris X. Lu, Peter ten Dijke  Molecular Cell  Volume 46, Issue 5, Pages 650-661 (June 2012) DOI: 10.1016/j.molcel.2012.04.003 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Identification of RNF12 as a Regulator of TGF-β Signaling (A) Functional screen with siRNAs that selectively depleted the expression of 5,000 druggable human genes using TGF-β-induced Smad3/4-driven reporter activity as readout. Among the screen results, locations of two siRNF12 are indicated together with control siRNAs (siALK5, siLuciferase, siSmad3). Testing, refers to other siRNAs that were analyzed. The X and Y axes (log base 2) are the relative luciferase activity, in two replicates. (B) RNF12 depletion strongly inhibits TGF-β-induced Smad2 phosphorylation and association with Smad4 in HaCaT cells. Two independent shRNAs (#1 and #2) were tested. Controls were treated with a control nontargeting shRNA. TGF-β (5 ng/ml) was added for 1, 2, or 6 hr. Lysed cells were subjected to anti-Smad2/3 immunoprecipitation and western blotting with indicated antibodies. (C) Ectopic expression of RNF12 enhances TGF-β-induced Smad2 phosphorylation and association with Smad4. (D) Reduced expression of TGF-β target genes: PAI-1, CTGF, Smad7, SnoN, p21, and p15 in stable RNF12-depleted HaCaT cells, assessed by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (E) RNF12 depletion in HaCaT cells inhibits TGF-β-induced growth inhibition as measured by cell counting. Control and RNF12 shRNA-expressing cells were stimulated with TGF-β for 1, 3, or 5 days, and then cells were counted. Data are presented as the mean ±SD. Co, control HaCaT cells infected with nontargeting shRNA. (F) Depletion of RNF12 mitigates TGF-β-induced EMT in HaCaT cells. Cells were treated with TGF-β for 36 hr. E-cadherin was visualized with an E-cadherin antibody (FITC). F-actin was stained with phalloidin (TRITC). (G) shRNA-mediated RNF12 depletion inhibits the TGF-β-induced expression of mesenchymal marker proteins, i.e., N-cadherin, fibronectin, and vimentin, as detected by western blotting. Co, control cells infected with nontargeting shRNA. Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 RNF12 Is a Smad7-Interacting Protein (A) RNF12 is a Smad7-interacting protein. HEK293T cells were transfected with Myc-tagged RNF12 CA and Flag-tagged Smad4 or Smad7. Cells were harvested for immunoprecipitation with anti-Flag affinity resin and immunoblotting with indicated antibodies. (B) RNF12 and Smad7 form an endogenous protein complex in mESCs, which is stabilized by MG132 (5 μM, 1 hr) treatment. (C) Ectopic expression of RNF12 by lentiviral infection rescues the RNF12/Smad7 interaction that was absent in Rnf12−/− mESCs. MG132 (5 μM) was added for 1 hr. (D) Ectopic expression of Smad7 by lentiviral infection rescues the RNF12/Smad7 interaction that was absent in Smad7−/− MEF cells. MG132 (5 μM) was added for 1 hr. Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 RNF12 Is a Smad7 E3 Ubiquitin Ligase In all ubiquitination assays, cells were pretreated with proteasome inhibitor. (A) shRNA-mediated RNF12 depletion by lentiviral infection decreases endogenous Smad7 polyubiquitination in HeLa cells. Note that Smad7 IP and Smad7 IB are shown in the right panel. (B) Ectopic expression of RNF12 by lentiviral infection rescues the ubiquitination of endogenous Smad7 in Rnf12−/− mESCs. (C) [35PS]-methionine labeling and pulse-chase studies of Smad7 in wild-type and Rnf12−/− mESCs. The amount of precipitated labeled protein after the chase is expressed as the percentage of the amount at the beginning of the chase (time 0). Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (D) Western analysis of V5-tagged Smad7 in infected wild-type and Rnf12−/− mESCs at the indicated intervals following cycloheximide treatment. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (E) RNF12 is required for TGF-β-induced Smad7 ubiquitination. Myc-ubiquitin-expressing HeLa cells were infected with control shRNA or shRNF12 lentivirus, and ubiquitinated Flag-Smad7 was visualized with an anti-Myc-ubiquitin antibody by western analysis after anti-Flag immunoprecipitation. (F) Western analysis of the TGF-β/Smad response in wild-type and Smad7−/− MEF cells with or without shRNA-mediated RNF12 depletion by lentiviral infection. Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Expression Levels of RNF12 Determine Activin Signaling Intensity in mESCs (A) Western blot analysis of RNF12, Smad7, and actin in mESCs at different time points during differentiation. (B) Comparative analysis of activin-induced Smad activity in Rnf12+/+ and Rnf12−/− mESCs transfected with the ARE-Luc transcriptional reporter. Data obtained from triplicates are presented as the mean ±SD. (C) Ectopic expression of RNF12 antagonizes the inhibitory effect of Smad7 on activin signaling in mESCs. Data from triplicates are presented as the mean ±SD of a representative experiment. (D) Smad7 expression levels increase and activin (Ac)-induced Smad2 phosphorylation decreases in Rnf12−/− mESCs. The basal levels of phosphorylated-Smad2 were inhibited by the addition of ALK4/5/7 kinase inhibitor SB431542 (10 μM). (E) Rnf12+/+ and Rnf12−/− mESCs were allowed to differentiate for 4 days on collagen IV-coated plates in the absence or presence of activin A. Expression levels of two activin-induced target genes, Lefty1 and Lefty2, were assessed by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (F) Morphological analysis of colonies of Rnf12+/+ and Rnf12−/− mESCs and wild-type mESCs infected with V5-Smad7 or V5-Smad7 and RNF12. Lower panel is AP staining of random single mES clone from upper panel. (G) Relative cell numbers of Rnf12+/+ and Rnf12−/− mESCs counted on day 5 and day 0. Each bar represents the mean ±SD (n = 3). (H) Ectopic expression of RNF12 antagonizes the growth-inhibitory effect of Smad7 on mESCs. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (I and J) Rnf12+/+ and Rnf12−/− mESCs were treated with or without activin A (50 ng/ml) or BMP2 (50 ng/ml) for 3 days. Relative cell numbers were compared between days 0 and 3. Each bar represents the mean ±SD (n = 3). Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 RNF12 Expression Levels Determine the Intensity of BMP/Smad Signaling (A) Deletion of RNF12 causes severe impairment of BMP-induced Smad1/5/8 -Smad4 interactions in mESCs. Rnf12+/+ and Rnf12−/− mESCs were treated with BMP2 or BMP type I receptor kinase inhibitor LDN-193189 (20 nM) for 1 hr before harvesting for immunoprecipitation and immunoblotting. (B) ChIP of BMP-stimulated Rnf12+/+ and Rnf12−/− mESCs performed with antibodies against Smad1/5/8 or phospho-Smad1/5/8. BMP-responsive regions of Id1 and an unresponsive control region of the same gene (control region) were analyzed by qRT-PCR. Data show the mean ±SD of triplicates and are representative of at least two independent experiments. (C) qRT-PCR analysis of genes directly targeted by BMP: Id1, Id3, and Smad6. Rnf12+/+ and Rnf12−/− mESCs were treated with BMP2 (50 ng/ml) for 1 hr before harvest. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 RNF12 Expression Levels Determine Activin- and BMP-Induced Differentiation Responses in mESCs (A) Activin A-induced anterior mesoderm differentiation is inhibited in RNF12-knockout mESCs. Expression of anterior mesoderm marker genes Goosecoid (Gsc) and LIM/homeobox protein 1 (Lhx1) was measured by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (B) (Left panel) Schematic representation of mESC differentiation under serum-free conditions with the addition of BMP2/4. Rnf12+/+ and Rnf12−/− mESCs were cultured in N2B27 supplemented serum-free media in the presence or absence of BMP2 and harvested 5 days later. Expression of the neural differentiation marker β-III tubulin (Tubb3) and Nestin was measured by qRT-PCR. No BMP2 treatment was set as “1.” Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Function of RNF12 in Zebrafish Embryos (A and B) Dose-dependent effects of overexpression of zRnf12. The phenotypes caused by zRnf12 overexpression included the following: type I, phenotype similar to wild-type embryo; type II, phenotype with a wider notochord and blood cell deposits; type III, phenotype with much more blood cells deposited. The red bracket represents the width of the notochord, and the white arrowhead indicates the deposited blood cells. The statistical values indicate the ratios of phenotypes in different groups of injected embryos. The underlined numbers indicate the total numbers of observed embryos. All embryos were observed at 25–26 hr postfertilization (hpf). (B) Dose-dependent induction of blood island caused by zRnf12 overexpression. (C) The expression patterns of no tail (ntl) and goosecoid (gsc) under conditions that caused zRnf12 knockdown (r12 MO) or/and squint (sqt) overexpression. All embryos are shown in the shield stage (middle gastrulation). All the embryo injections were carefully controlled with the same amount of control MO and control GFP mRNA. (D) (Da) The most common phenotype in zebrafish embryos injected with smad7 mRNA (200 pg); this phenotype was called “Sphere.” (Db) The phenotype caused by injecting zRnf12 mRNA. (Dc) Major phenotype in zebrafish embryos coinjected with smad7 and zRnf12 mRNAs; this phenotype was called “Little Tail.” (Dd) The statistical ratios of phenotypes in embryos injected with different mRNAs and their combinations. All embryos were observed at 31 hpf with the head oriented to the left. The number of embryos showing the phenotype was indicated. (E) The shh expression pattern of the same types of embryos shown in (Da)–(Dc); (upper panel) the lateral view with the head oriented to the left; (lower panel) the dorsal view with the head oriented to the left. All embryos were fixed at 24 hpf. The number of embryos showing the phenotype is indicated. (F) Relative shh expression levels of the same types of embryos shown in (Da)–(Dc) as detected by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (G) Relative ntl expression levels of the same types of embryos shown in (Da)–(Dc) as detected by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments. (H and I) Western blotting for Smad7-GFP fusion protein levels affected by zRnf12 MO or ectopic expression of zrnf12. (J) Western blotting for endogenous Smad1/5/8 and Smad2 C-terminal phosphorylation levels affected by zrnf12 MO or zRnf12mRNA. (K) The knockdown of zebrafish smad7 can rescue rnf12 morphants phenotype. The statistical bars are listed on the right. All phenotypes were monitored at 22–24 hpf. Arrow indicated bigger head and anterior phenotype caused by rnf12 MO; Fused-So indicated fused-somite defect caused by smad7 MO. The injection dose for every embryo was 4 ng of the MO mixture (2 ng of MO1 and 2 ng MO2 pooled). Molecular Cell 2012 46, 650-661DOI: (10.1016/j.molcel.2012.04.003) Copyright © 2012 Elsevier Inc. Terms and Conditions