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• W2010417259 abstract "BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates. BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates. Embryonic stem cells (ESCs) 3The abbreviations used are: ESCembryonic stem cellBMPbone morphogenic proteinBREBMP responsive elementChIPchromatin immunoprecipitationCochcochlinGFPgreen fluorescence proteinLIFleukemia inhibitory factorshRNAshort hairpin RNAAPalkaline phosphatase. are pluripotent stem cells that can indefinitely self-renew in vitro while at the same time possess the ability to differentiate into all the adult tissues, including germ cells (1Wobus A.M. Boheler K.R. Embryonic stem cells: prospects for developmental biology and cell therapy.Physiol. Rev. 2005; 85: 635-678Crossref PubMed Scopus (593) Google Scholar, 2Young R.A. Control of the embryonic stem cell state.Cell. 2011; 144: 940-954Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). Because of two remarkable properties (self-renewal and differentiation), ESCs are valuable for a wide range of applications, such as an in vitro model to study early embryo development, and most importantly, as a promising cell source for transplantation in regenerative medicine (1Wobus A.M. Boheler K.R. Embryonic stem cells: prospects for developmental biology and cell therapy.Physiol. Rev. 2005; 85: 635-678Crossref PubMed Scopus (593) Google Scholar, 2Young R.A. Control of the embryonic stem cell state.Cell. 2011; 144: 940-954Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 3Evans M.J. Kaufman M.H. Establishment in culture of pluripotential cells from mouse embryos.Nature. 1981; 292: 154-156Crossref PubMed Scopus (6423) Google Scholar, 4Martin G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 7634-7638Crossref PubMed Scopus (4288) Google Scholar). It has been recognized that mouse ESC fate determination is delicately regulated by multiple internal transcription factors and external signaling pathways. Internal core transcription factors, such as OCT4/POU5F1, NANOG, and SOX2, can form an autoregulatory feedback circuit to maintain pluripotency of ESCs. These factors physically interact with each other and co-occupy the promoters of target genes to establish ESC-specific transcriptome (2Young R.A. Control of the embryonic stem cell state.Cell. 2011; 144: 940-954Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 5Cole M.F. Young R.A. Mapping key features of transcriptional regulatory circuitry in embryonic stem cells.Cold Spring Harb Symp Quant Biol. 2008; 73: 183-193Crossref PubMed Scopus (30) Google Scholar). Several epigenetic factors controlling histone modification and chromatin structures also play important roles during the switch between self-renewal and differentiation of ESCs (6Bibikova M. Laurent L.C. Ren B. Loring J.F. Fan J.B. Unraveling epigenetic regulation in embryonic stem cells.Cell Stem Cell. 2008; 2: 123-134Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 7Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. Bell G.W. Otte A.P. Vidal M. Gifford D.K. Young R.A. Jaenisch R. Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature. 2006; 441: 349-353Crossref PubMed Scopus (2035) Google Scholar, 8Lee T.I. Jenner R.G. Boyer L.A. Guenther M.G. Levine S.S. Kumar R.M. Chevalier B. Johnstone S.E. Cole M.F. Isono K. Koseki H. Fuchikami T. Abe K. Murray H.L. Zucker J.P. Yuan B. Bell G.W. Herbolsheimer E. Hannett N.M. Sun K. Odom D.T. Otte A.P. Volkert T.L. Bartel D.P. Melton D.A. Gifford D.K. Jaenisch R. Young R.A. Control of developmental regulators by Polycomb in human embryonic stem cells.Cell. 2006; 125: 301-313Abstract Full Text Full Text PDF PubMed Scopus (1866) Google Scholar, 9Christophersen N.S. Helin K. Epigenetic control of embryonic stem cell fate.J. Exp. Med. 2010; 207: 2287-2295Crossref PubMed Scopus (105) Google Scholar). Appropriate activity of extracellular signal-regulated kinase (ERK) within the cells also plays a central role in the fate choices of mouse ESCs (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (2528) Google Scholar, 12Li Z. Chen Y.G. Fine-tune of intrinsic ERK activity by extrinsic BMP signaling in mouse embryonic stem cells.Protein Cell. 2012; 3: 401-404Crossref PubMed Scopus (7) Google Scholar). Besides these intrinsic factors, extracellular cytokines such as leukemia inhibitory factor (LIF), transforming growth factor β (TGF-β) superfamily, and Wnt signaling have also been shown to govern ESC fate (13Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1725) Google Scholar, 14Williams R.L. Hilton D.J. Pease S. Willson T.A. Stewart C.L. Gearing D.P. Wagner E.F. Metcalf D. Nicola N.A. Gough N.M. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.Nature. 1988; 336: 684-687Crossref PubMed Scopus (1528) Google Scholar, 15Ying Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar). Among them, LIF signaling has long been established to support mouse ESC self-renewal on standard serum- and feeder-dependent culture conditions (16Niwa H. Burdon T. Chambers I. Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1245) Google Scholar). LIF transduces its signal through binding to a heterodimeric receptor complex consisting of gp130 and the low-affinity LIF receptor and then activates STAT3 through phosphorylation (15Ying Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar, 16Niwa H. Burdon T. Chambers I. Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1245) Google Scholar). However, LIF alone is not enough to maintain mouse ESC pluripotency, and the cooperation between BMP4 and LIF is found to be sufficient for long-term self-renewal maintenance in the absence of serum and feeder cells (11Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (2528) Google Scholar, 15Ying Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar). embryonic stem cell bone morphogenic protein BMP responsive element chromatin immunoprecipitation cochlin green fluorescence protein leukemia inhibitory factor short hairpin RNA alkaline phosphatase. BMP4 is a TGF-β superfamily member, which plays multiple critical roles during embryogenesis as well as in tissue homeostasis by regulating a series of cellular processes including cell proliferation, adhesion, migration, differentiation, and apoptosis (17Massagué J. Chen Y.G. Controlling TGF-β signaling.Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 18Shi Y. Massagué J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4794) Google Scholar, 19Fei T. Chen Y.G. Regulation of embryonic stem cell self-renewal and differentiation by TGF-β family signaling.Sci. China Life Sci. 2010; 53: 497-503Crossref PubMed Scopus (24) Google Scholar). BMPs transduce their signal through binding to the transmembrane type I and type II serine/threonine kinase receptor complexes. The ligand-activated receptors then phosphorylate and activate R-Smads (Smad1, -5, and -8), leading to the formation of a complex consisting of R-Smad and Co-Smad (Smad4). Translocation of the Smad complex from cytoplasm to the nucleus enables direct transcriptional regulation of target gene expression (17Massagué J. Chen Y.G. Controlling TGF-β signaling.Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 20Massagué J. TGF-β signal transduction.Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3981) Google Scholar, 21Feng X.H. Derynck R. Specificity and versatility in TGF-β signaling through Smads.Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1528) Google Scholar, 22Massagué J. Seoane J. Wotton D. Smad transcription factors.Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1923) Google Scholar). Considering the importance of BMP signaling in mouse ESC fate control, it is of great value to learn how BMP signaling exerts its functions. ID family proteins were first described as critical downstream targets of BMP4 to promote self-renewal by inhibiting neural differentiation (15Ying Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar). Our previous work extended the repertoire of potential direct target genes of BMP signaling in mouse ESCs from a genome-wide perspective (23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar) and further identified another important direct target gene, Dusp9, which mediates BMP signaling to balance ERK activity (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the present study, we report the identification of cochlin (coagulation factor C homolog) (Coch), as another important direct target gene of BMP signaling, which promotes mouse ESC self-renewal by inhibition of neural differentiation. Feeder-free E14 and R1 mouse ES cells were grown on gelatin-coated dishes and cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 15% Knockout Serum Replacement (KSR) (Invitrogen), 2 mm glutamine, 10−4 m nonessential amino acids, 10−4 m β-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1,000 units/ml mouse LIF. To examine the effect of Coch on the self-renewal of mouse ESCs, R1 cells were cultured in N2B27 medium containing LIF or LIF plus BMP. For neural differentiation, R1 cells were cultured in N2B27 medium without any cytokines, as described previously (23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). NMuMG, NIH3T3, C2C12, and HeLa cells were cultured in DMEM supplemented with 10% FBS (Hyclone). BMP4 was purchased from R&D. The antibodies used in this study are listed as follows: rabbit anti-Smad4 antibody was generated from human Smad4-linker (aa144–316)-His; Smad1 (sc-7965X) was purchased from Santa Cruz Biotechnology; anti-nestin antibody (MAB353) was purchased from Chemicon International. Mouse Coch (NC_000078) promoter constructs (−2609 bp ∼ +137 bp) were generated by PCR using mouse genomic DNA as a template and amplified fragments were cloned into the pGL3-basic reporter vector through MluI/BglII sites. A series of 5′ deletion constructs were generated using the longest Coch (−2609 bp ∼ +137 bp) fragment as the PCR template, and the PCR primers used are listed below: −2609 bp ∼ +137 bp construct: 5′-GTTACGCGTAGAACCAGCACCTTTCTG-3′ (sense); −2010 bp ∼ +137 bp construct: 5′-GCTACGCGTGACTCCTCCACCCTCCTT-3′ (sense); −1774 bp ∼ +137 bp construct: 5′-GTTACGCGTGACCATGCTGCCTGAGTT-3′ (sense); −1360 bp ∼ +137 bp construct: 5′-GTTACGCGTGCTGACGCACACTTCAGC-3′ (sense); −640 bp ∼ +137 bp construct: 5′-GTTACGCGTCAACAAATGACCCATCAG-3′ (sense); −481 bp ∼ +137 bp construct: 5′-GTTACGCGTTCTCAGAACCGGGTGGCT-3′ (sense); −320 bp ∼ +137 bp construct: 5′-GTTACGCGTCACCACAGGCGACCTTCA-3′ (sense); −36 bp ∼ +137 bp construct: 5′-GTTACGCGTGGAGGTGCATAGGGTCTG-3′ (sense). All of the above constructs were generated using the same antisense primer: 5′-GACAGATCTAGGCGTGAACGAGGTTCG-3′ (antisense). The −320 bp ∼ −57 bp construct was generated from −320 bp ∼ +137 bp construct by digesting the template with MluI and BamHI enzymes. Site-directed mutagenesis was performed using −320 bp ∼ −57 bp construct as a template. The wild-type GGCGCC sites were replaced by the mutated GaattC-mutation sequence (Fig. 2D). For overexpression of Coch, the coding sequence (CDS) of full-length mouse Coch with or without HA (hemagglutinin) tag (named COCH-1 and COCH-2, respectively) were cloned into pENTR1A backbone and then using LR clonase reactions (Invitrogen) to replace these CDS into the p2k7neo lentiviral vector under the control of EF1α promoter (24Kee K. Angeles V.T. Flores M. Nguyen H.N. Reijo Pera R.A. Human DAZL, DAZ, and BOULE genes modulate primordial germ-cell and haploid gamete formation.Nature. 2009; 462: 222-225Crossref PubMed Scopus (404) Google Scholar). The short hairpin RNA (shRNA) targeting mouse Coch mRNA (NM_007728) or the control firefly luciferase gene was cloned into the pll3.7 lentiviral vector, which was modified with GFP region replaced by puromycin gene. The sequences of the shRNAs targeting mouse Coch are listed below: shCoch-1: 5′-CAGGGTTAGAACCAATTGT-3′; shCoch-2: 5′-GCCGATTTAATTTGCAGAA-3′. The shSmad1 and shSmad4 plasmids were generated previously (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). Lentivirus was produced by transfection of HEK293FT cells with lentiviral vectors, which contain the CDS or shRNA sequences of Coch using Lipofectamine 2000 (Invitrogen). After 3 days, the supernatant was collected and filtered through a 0.45 μm filter (Millipore). Virus-containing supernatants were added into plates of ES cells in the presence of 8 μg/ml polybrene. After incubation for 6 h at 37 °C, equal volume of complete mouse ESC culture medium (DMEM supplemented with 15% KSR, l-glutamine, nonessential amino acids, β-mercaptoethanol, and 1,000 units/ml LIF) was added and further incubated overnight. Then the supernatants were discarded, and cells were washed three or four times with PBS. Then the cells can be cultured with the complete ESC medium. In this study, Geneticin (250 μg/ml, Invitrogen) and puromycin (1000 μg/ml) were added into culture medium for 3 to 5 days to select cells with stable viral integration. Cells were seeded in 48-well plate and transfected with the reporter constructs when they reach to 30–50% confluent. pRenilla-TK vector was transfected as an internal control. Transfection was carried out with Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. One day after transfection, 10 ng/ml BMP4 was added into the medium. Cells were lysed after BMP4 treatment for 24 h and then subjected to luciferase activity measurement. Each experiment was done in triplicate, and all the data are normalized to Renilla activity. The data were represented as the mean ± S.E. of three independent experiments. Real-time PCR was performed as described previously (23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). Briefly, the total RNA was isolated from the cells using TRIzol reagent (Invitrogen). Complementary DNA synthesis was primed with oligo dT (Takara) by reverse transcriptase (Toyobo). Real-time PCR was carried out using EvaGreen dye (Biotium) on the Mx3000P system. All the primers used are listed in supplemental Table S1. Alkaline phosphatase staining was performed using Alkaline Phosphatase Detection Kit (SCR004, Millipore) according to the manufacturer's instruction. Immunostaining and Western blot were carried out as described previously (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). ChIP was carried out as described previously (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). Briefly, cells were firstly cross-linked by 1% formaldehyde solution for 15 min. Then the cells were lysed with lysis buffer (1% SDS, 50 mm Tris pH 8.0, 5 mm EDTA, proteinase inhibitors) and sonicated to get DNA fragments ranging ∼300–500 bp on average. After centrifuging at 14,000 rpm for 10 min, the supernatant was collected and pre-absorbed by 50 μl of protein A beads (Zymed Laboratories Inc.). After incubation with 10 μg of antibodies (control IgG, anti-Smad1, and anti-Smad4) overnight at 4 °C, the immunocomplexes were collected with 50 μl of protein A beads by 3 h co-incubation and then washed sequentially with salt buffers. The bound immunocomplexes were eluted with elution buffer (25 mm Tris, pH 8.0, 10 mm EDTA, 0.5% SDS) by heating at 65 °C with occasional vortex for 15 min and crosslinking was reversed by overnight incubation at 65 °C. Immunoprecipitated DNA and input DNA were then purified by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation after treatment with RNase A and proteinase K. ChIP primers used in this study are listed in the supplemental Table S1. To identify direct downstream target genes of BMP signaling, we previously carried out Smad ChIP-chip and ChIP-seq combined with RNA expression microarray upon BMP4 stimulation in mouse ESCs (23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). In addition to the previously characterized Id family genes (15Ying Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar), Dusp9 (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), Dypsl2, and Kdm6b (23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar), another gene named Coch exhibited significant up-regulation (2.1-fold change) in the RNA expression microarray data in response to BMP4 stimulation (10Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. Zhang H. Han J.D. Chen Y.G. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Fei T. Xia K. Li Z. Zhou B. Zhu S. Chen H. Zhang J. Chen Z. Xiao H. Han J.D. Chen Y.G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.Genome Res. 2010; 20: 36-44Crossref PubMed Scopus (99) Google Scholar). We confirmed this up-regulation by quantitative real-time PCR (qRT-PCR), and a dramatic up-regulation was observed (Fig. 1A). BMP4 treatment steadily induced Coch mRNA expression in E14 mouse ESCs, and the induction reached more than 12-fold after 24 h (Fig. 1A). Similar up-regulation could also be observed in R1 mouse ESCs (Fig. 1B), indicating Coch is a bona fide BMP target gene in mouse ESCs. To examine the cell type specificity of Coch up-regulation by BMP4, we treated differentiated mouse somatic cells NMuMG, NIH3T3, and C2C12 cells with 10 ng/ml BMP4 for 8 h. As shown in Fig. 1B, Coch did not exhibit any significant change while the classic BMP target gene, Id1, was consistently up-regulated in these cells. Thus, the up-regulation of Coch by BMP4 was specific to mouse ESCs. In addition, we also found that human COCH is up-regulated by BMP4 in H1 human ESCs (Fig. 1C). Consistent with the results from mouse cells, human COCH did not respond to BMP4 in differentiated human somatic cells (Hep3B and HepG2), where ID1 was up-regulated by BMP4 (Fig. 1D). These data collectively indicate that BMP4 can up-regulate Coch expression, specifically in ESCs. BMP/Smad signaling regulates its downstream target genes through Smad1/5/8 and Smad4 (22Massagué J. Seoane J. Wotton D. Smad transcription factors.Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1923) Google Scholar). To examine whether induction of Coch by BMP4 was dependent on Smad proteins, we knocked down Smad1 as well as Smad4 in wild-type and Smad5−/− TC-1 mouse ESCs. As is shown in Fig. 2A, either Smad1 or Smad4 knockdown attenuated the up-regulation of Coch by BMP4, and the most dramatic attenuation of Coch induction was observed in Smad5 knockout ESCs with Smad4 knockdown, indicating that Smad1, Smad5, and Smad4 are responsible for BMP induction of Coch. The residual induction of Coch after shSmad1 in Smad5 knockout cells might result from incomplete deletion of Smad1. These data indicate that the induction of Coch by BMP4 is dependent on Smad1/5 and Smad4. As Smad1 and Smad4 are transcription factors, they regulate the expression of downstream target genes by binding to specific DNA elements, such as CAGAC, GCCGnCGC, GGCGCC in gene promoters (25Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. Direct binding of Smad3 and Smad4 to critical TGFβ-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1580) Google Scholar, 26Jonk L.J. Itoh S. Heldin C.H. ten Dijke P. Kruijer W. Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-β, activin, and bone morphogenetic protein-inducible enhancer.J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 27Kusanagi K. Inoue H. Ishidou Y. Mishima H.K. Kawabata M. Miyazono K. Characterization of a bone morphogenetic protein-responsive Smad-binding element.Mol. Biol. Cell. 2000; 11: 555-565Crossref PubMed Scopus (155) Google Scholar, 28Ishida W. Hamamoto T. Kusanagi K. Yagi K. Kawabata M. Takehara K. Sampath T.K. Kato M. Miyazono K. Smad6 is a Smad1/5-induced smad inhibitor. Characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter.J. Biol. Chem. 2000; 275: 6075-6079Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 29Korchynskyi O. ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter.J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Therefore, we attempted to identify the BMP responsive element (BRE) in the Coch promoter. To this end, we cloned putative mouse Coch promoter covering the −2609 base pair (bp) to +137 bp around the transcriptional start site (TSS) and inserted it into pGL3-basic luciferase reporter plasmid. Reporter assay showed that it responded to BMP4 stimulation by about 2-fold (Fig. 2B). Then, a series of deletion constructs were generated and tested for BMP4 responsiveness. As shown in Fig. 2B, the −1360 bp ∼ −36 bp region still retained the intact response to BMP. Further deletion within −1360 bp ∼ −36 bp region showed that two subregions (−481 bp ∼ −320 bp and −320 bp ∼ −57 bp) contained the minimal BREs for Coch up-regulation. The fragment of −320 bp ∼ −57 bp still retained partial response to BMP4 (Fig. 2, B and C). By scrutinizing the potential Smad binding elements within the −320 bp ∼ −57 bp region, we found two putative GC-rich BREs (BRE1 and BRE2), and mutation of key nucleotides within BRE1 but not in BRE2 totally abolished the transcription activation by BMP4 (Fig. 2, C and D), indicating that BRE1 is necessary for Coch up-regulation by BMP4. Further analysis of −480 bp to −320 bp region by either the region alone or mutation analysis of putative BREs in that region using reporter assay excluded the possibility that there are other functional BREs in that region (data not shown). To examine whether Smad1/5 and Smad4 could physically bind to this region of Coch promoter upon BMP4 treatment, we conducted a ChIP assay using Smad1 and Smad4 antibodies. Five pairs of primers were designed to amplify the putative Smad binding region along the Coch promoter (supplemental Table S1). Only primer pair #4, whose amplicon covers the BRE1 region identified above, showed a binding signal for both Smad1 and Smad4 in response to BMP4 treatment (Fig. 3A). In addition, by comparing the putative Coch promoter region (3000 bp upstream o" @default.
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• W2010417259 date "2013-03-01" @default.
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• W2010417259 title "BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells" @default.
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