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Robert C. Angerer - One of the best experts on this subject based on the ideXlab platform.
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spatially regulated spets4 transcription factor activity along the sea urchin Embryo Animal vegetal axis
Development, 1999Co-Authors: Lynne M. Angerer, Robert C. AngererAbstract:Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined Animal-vegetal axis of the very early sea urchin Embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity. Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L. M. and Angerer, R. C. (1997) Dev. Biol. 187, 71–78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R. C. and Angerer, L. M. (1999) Mol. Cell. Biol. 19, 1271–1278). Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription. The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the Embryo during late cleavage/early blastula stages. Although maternal transcripts are uniformly distributed in the egg and early cleaving Embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription. In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a beta-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the Animal-vegetal axis of the sea urchin Embryo.
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multiple positive cis elements regulate the asymmetric expression of the sphe gene along the sea urchin Embryo Animal vegetal axis
Developmental Biology, 1997Co-Authors: Lynne M. Angerer, Robert C. AngererAbstract:Abstract The mechanism that establishes the maternally determined Animal–vegetal axis of sea urchin Embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early Embryo.
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Multiple Positive cis Elements Regulate the Asymmetric Expression of the SpHE Gene along the Sea Urchin Embryo Animal-Vegetal Axis
Developmental biology, 1997Co-Authors: Zheng Wei, Lynne M. Angerer, Robert C. AngererAbstract:Abstract The mechanism that establishes the maternally determined Animal–vegetal axis of sea urchin Embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early Embryo.
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Fate Specification Along the Sea Urchin Embryo Animal-Vegetal Axis
The Biological bulletin, 1997Co-Authors: Robert C. Angerer, Lynne M. AngererAbstract:ROBERT C. ANGERER AND LYNNE M. ANGERER Department QfBiology, University afRochester, Rochester, New York 14627 Introduction Like those of a large majority of taxa, sea urchin em- bryos establish a spatial coordinate system for the initial body plan from one axis, the Animal-vegetal (A-V), that is fixed during oogenesis by asymmetric deposition of maternal molecules (the Embryologists’ “determinants”) and a second axis, dorsal-ventral (or, more descriptively, oral-aboral), that is specified sometime during the first few cleavage divisions (reviewed by Davidson, 1989). The ability of sea urchin Embryos to establish these axes while continuously reorienting in culture suggests that neither axis is sensitive to the earth’s gravitational field. In Embryos of many sea urchin species, A-V polarity is evidenced by the unequal sizes of blastomeres of the 16- cell Embryo, which consists of tiers of eight mesomeres, four macromeres, and four micromeres. Classical exper- imental micromanipulations of Embryos (reviewed by Horstadius, 1973) have established that the fates of mi- cromeres are determined by inheritance of maternal molecules. In addition, the micromeres provide a vegetal focus of inductive influence that is critical in the normal Embryo for appropriate specification of fates of overlying Animal blastomeres, and that can induce vegetal differ- entiation (gut, secondary mesenchyme) in cells of more Animal tiers when micromeres are transplanted to ec- topic sites (Khaner and Wilt, 199 1; Ransick and David- son, 1993). Thus, specification of fates along the AV axis utilizes both major mechanisms familiar to developmen- tal biologists-inheritance of maternally provided posi-
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The SpHE gene is downregulated in sea urchin late blastulae despite persistence of multiple positive factors sufficient to activate its promoter
Mechanisms of development, 1997Co-Authors: Zheng Wei, Lynne M. Angerer, Alan P. Kenny, Robert C. AngererAbstract:Abstract Previous studies of the regulatory region of the SpHE (hatching enzyme) gene of the sea urchin Strongylocentrotus purpuratus (Wei, Z., Angerer, L.M., Gagnon, M.L. and Angerer, R.C. (1995) Characterization of the SpHE promoter that are spatially regulated along the Animal-vegetal axis of the sea urchin Embryo. Dev. Biol. 171, 195–211) have shown that approximately 330 bp is necessary and sufficient to promote high level expression in Embryos of transgenes that reproduce the spatially asymmetric pattern of endogenous gene activity along the maternally determined Animal-vegetal Embryonic axis. Furthermore, SpHE regulatory elements appear to be redundant since several different combinations are sufficient to elicit strong promoter activity and many subsets function like the endogenous gene only in non-vegetal cells of the blastula (Wei, Z., Angerer, L.M. and Angerer, R.C. (1997) Multiple positive cis -elements regulate the asymmetric expression of the SpHE gene along the sea urchin Embryo Animal-vegetal axis. Dev. Biol., 187, 71–88). Here we demonstrate by in vivo footprinting that many cis elements on the endogenous promoter are occupied when the gene is active in early blastulae, but the binding of corresponding trans factors is significantly reduced when the gene becomes inactive in late blastulae. In addition, downregulation of the promoter is accompanied by a transition from a non-nucleosomal to a nucleosome-like chromatin structure. Surprisingly, in vitro DNase I footprints of the 300 bp promoter using nuclear protein extracts from early and late blastulae are not detectably different and neither this sequence, nor a longer one extending to −1255, reproduces the loss of endogenous SpHE transcriptional activity after very early blastula stage. These observations imply that temporal repression of SpHE transcription involves a decrease in accessibility of the promoter to activators that are nevertheless present in nuclei and capable of activating transgene promoters. Temporal, but not spatial, downregulation is therefore likely to be regulated by negative activities functioning outside the −1255 promoter region which may serve as direct repressors or mediate an inactive chromatin structure.
Lynne M. Angerer - One of the best experts on this subject based on the ideXlab platform.
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spatially regulated spets4 transcription factor activity along the sea urchin Embryo Animal vegetal axis
Development, 1999Co-Authors: Lynne M. Angerer, Robert C. AngererAbstract:Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined Animal-vegetal axis of the very early sea urchin Embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity. Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L. M. and Angerer, R. C. (1997) Dev. Biol. 187, 71–78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R. C. and Angerer, L. M. (1999) Mol. Cell. Biol. 19, 1271–1278). Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription. The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the Embryo during late cleavage/early blastula stages. Although maternal transcripts are uniformly distributed in the egg and early cleaving Embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription. In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a beta-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the Animal-vegetal axis of the sea urchin Embryo.
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multiple positive cis elements regulate the asymmetric expression of the sphe gene along the sea urchin Embryo Animal vegetal axis
Developmental Biology, 1997Co-Authors: Lynne M. Angerer, Robert C. AngererAbstract:Abstract The mechanism that establishes the maternally determined Animal–vegetal axis of sea urchin Embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early Embryo.
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Multiple Positive cis Elements Regulate the Asymmetric Expression of the SpHE Gene along the Sea Urchin Embryo Animal-Vegetal Axis
Developmental biology, 1997Co-Authors: Zheng Wei, Lynne M. Angerer, Robert C. AngererAbstract:Abstract The mechanism that establishes the maternally determined Animal–vegetal axis of sea urchin Embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early Embryo.
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Fate Specification Along the Sea Urchin Embryo Animal-Vegetal Axis
The Biological bulletin, 1997Co-Authors: Robert C. Angerer, Lynne M. AngererAbstract:ROBERT C. ANGERER AND LYNNE M. ANGERER Department QfBiology, University afRochester, Rochester, New York 14627 Introduction Like those of a large majority of taxa, sea urchin em- bryos establish a spatial coordinate system for the initial body plan from one axis, the Animal-vegetal (A-V), that is fixed during oogenesis by asymmetric deposition of maternal molecules (the Embryologists’ “determinants”) and a second axis, dorsal-ventral (or, more descriptively, oral-aboral), that is specified sometime during the first few cleavage divisions (reviewed by Davidson, 1989). The ability of sea urchin Embryos to establish these axes while continuously reorienting in culture suggests that neither axis is sensitive to the earth’s gravitational field. In Embryos of many sea urchin species, A-V polarity is evidenced by the unequal sizes of blastomeres of the 16- cell Embryo, which consists of tiers of eight mesomeres, four macromeres, and four micromeres. Classical exper- imental micromanipulations of Embryos (reviewed by Horstadius, 1973) have established that the fates of mi- cromeres are determined by inheritance of maternal molecules. In addition, the micromeres provide a vegetal focus of inductive influence that is critical in the normal Embryo for appropriate specification of fates of overlying Animal blastomeres, and that can induce vegetal differ- entiation (gut, secondary mesenchyme) in cells of more Animal tiers when micromeres are transplanted to ec- topic sites (Khaner and Wilt, 199 1; Ransick and David- son, 1993). Thus, specification of fates along the AV axis utilizes both major mechanisms familiar to developmen- tal biologists-inheritance of maternally provided posi-
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The SpHE gene is downregulated in sea urchin late blastulae despite persistence of multiple positive factors sufficient to activate its promoter
Mechanisms of development, 1997Co-Authors: Zheng Wei, Lynne M. Angerer, Alan P. Kenny, Robert C. AngererAbstract:Abstract Previous studies of the regulatory region of the SpHE (hatching enzyme) gene of the sea urchin Strongylocentrotus purpuratus (Wei, Z., Angerer, L.M., Gagnon, M.L. and Angerer, R.C. (1995) Characterization of the SpHE promoter that are spatially regulated along the Animal-vegetal axis of the sea urchin Embryo. Dev. Biol. 171, 195–211) have shown that approximately 330 bp is necessary and sufficient to promote high level expression in Embryos of transgenes that reproduce the spatially asymmetric pattern of endogenous gene activity along the maternally determined Animal-vegetal Embryonic axis. Furthermore, SpHE regulatory elements appear to be redundant since several different combinations are sufficient to elicit strong promoter activity and many subsets function like the endogenous gene only in non-vegetal cells of the blastula (Wei, Z., Angerer, L.M. and Angerer, R.C. (1997) Multiple positive cis -elements regulate the asymmetric expression of the SpHE gene along the sea urchin Embryo Animal-vegetal axis. Dev. Biol., 187, 71–88). Here we demonstrate by in vivo footprinting that many cis elements on the endogenous promoter are occupied when the gene is active in early blastulae, but the binding of corresponding trans factors is significantly reduced when the gene becomes inactive in late blastulae. In addition, downregulation of the promoter is accompanied by a transition from a non-nucleosomal to a nucleosome-like chromatin structure. Surprisingly, in vitro DNase I footprints of the 300 bp promoter using nuclear protein extracts from early and late blastulae are not detectably different and neither this sequence, nor a longer one extending to −1255, reproduces the loss of endogenous SpHE transcriptional activity after very early blastula stage. These observations imply that temporal repression of SpHE transcription involves a decrease in accessibility of the promoter to activators that are nevertheless present in nuclei and capable of activating transgene promoters. Temporal, but not spatial, downregulation is therefore likely to be regulated by negative activities functioning outside the −1255 promoter region which may serve as direct repressors or mediate an inactive chromatin structure.
Joan Massague - One of the best experts on this subject based on the ideXlab platform.
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a human mad protein acting as a bmp regulated transcriptional activator
Nature, 1996Co-Authors: Fang Liu, Akiko Hata, Julie C Baker, Jacqueline Doody, Juan M Carcamo, Richard M Harland, Joan MassagueAbstract:The TGF-beta/activin/BMP cytokine family signals through serine/threonine kinase receptors, but how the receptors transduce the signal is unknown. The Mad (Mothers against decapentaplegic) gene from Drosophila and the related Sma genes from Caenorhabditis elegans have been genetically implicated in signalling by members of the bone-morphogenetic-protein (BMP) subfamily. We have cloned Smad1, a human homologue of Mad and Sma. Microinjection of Smad1 messenger RNA into Xenopus Embryo Animal caps mimics the mesoderm-ventralizing effects of BMP4. Smad1 moves into the nucleus in response to BMP4. Smad1 has transcriptional activity when fused to a heterologous DNA-binding domain, and this activity is increased by BMP4 acting through BMP-receptor types I and II. The transactivating activity resides in the conserved carboxy-terminal domain of Smad1 and is disrupted by a nonsense mutation that corresponds to null mutations found in Mad and in the related gene DPC4, a candidate tumour-suppressor gene in human pancreatic cancer. Additionally, we show that DPC4 contains a transcriptional activation domain. The results suggests that the Smad proteins are a new class of transcription factors that mediate responses to the TGF-beta family.
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A human Mad protein acting as a BMP-regulated transcriptional activator
Nature, 1996Co-Authors: Fang Liu, Akiko Hata, Julie C Baker, Jacqueline Doody, Juan M Carcamo, Richard M Harland, Joan MassagueAbstract:THE TGF-β/activin/BMP cytokine family signals through serine/threonine kinase receptors, but how the receptors transduce the signal is unknown. The Mad (Mothers against decapentaplegic) gene from Drosophila ^1 and the related Sma genes from Caenorhabditis elegans ^2 have been genetically implicated in signalling by members of the bone-morphogenetic-protein (BMP) subfamily. We have cloned Smad1 , a human homologue of Mad and Sma . Microinjection of Smad1 messenger RNA into Xenopus Embryo Animal caps mimics the mesoderm-ventralizing effects of BMP4. Smad1 moves into the nucleus in response to BMP4. Smad1 has transcriptional activity when fused to a heterologous DNA-binding domain, and this activity is increased by BMP4 acting through BMP-receptor types I and II. The transactivating activity resides in the conserved carboxy-terminal domain of Smad1 and is disrupted by a nonsense mutation that corresponds to null mutations found in Mad and in the related gene DPC4 , a candidate tumour-suppressor gene in human pancreatic cancer^3. Additionally, we show that DPC4 contains a transcriptional activation domain. The results suggests that the Smad proteins are a new class of transcription factors that mediate responses to the TGF-β family.
Zheng Wei - One of the best experts on this subject based on the ideXlab platform.
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Multiple Positive cis Elements Regulate the Asymmetric Expression of the SpHE Gene along the Sea Urchin Embryo Animal-Vegetal Axis
Developmental biology, 1997Co-Authors: Zheng Wei, Lynne M. Angerer, Robert C. AngererAbstract:Abstract The mechanism that establishes the maternally determined Animal–vegetal axis of sea urchin Embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early Embryo.
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The SpHE gene is downregulated in sea urchin late blastulae despite persistence of multiple positive factors sufficient to activate its promoter
Mechanisms of development, 1997Co-Authors: Zheng Wei, Lynne M. Angerer, Alan P. Kenny, Robert C. AngererAbstract:Abstract Previous studies of the regulatory region of the SpHE (hatching enzyme) gene of the sea urchin Strongylocentrotus purpuratus (Wei, Z., Angerer, L.M., Gagnon, M.L. and Angerer, R.C. (1995) Characterization of the SpHE promoter that are spatially regulated along the Animal-vegetal axis of the sea urchin Embryo. Dev. Biol. 171, 195–211) have shown that approximately 330 bp is necessary and sufficient to promote high level expression in Embryos of transgenes that reproduce the spatially asymmetric pattern of endogenous gene activity along the maternally determined Animal-vegetal Embryonic axis. Furthermore, SpHE regulatory elements appear to be redundant since several different combinations are sufficient to elicit strong promoter activity and many subsets function like the endogenous gene only in non-vegetal cells of the blastula (Wei, Z., Angerer, L.M. and Angerer, R.C. (1997) Multiple positive cis -elements regulate the asymmetric expression of the SpHE gene along the sea urchin Embryo Animal-vegetal axis. Dev. Biol., 187, 71–88). Here we demonstrate by in vivo footprinting that many cis elements on the endogenous promoter are occupied when the gene is active in early blastulae, but the binding of corresponding trans factors is significantly reduced when the gene becomes inactive in late blastulae. In addition, downregulation of the promoter is accompanied by a transition from a non-nucleosomal to a nucleosome-like chromatin structure. Surprisingly, in vitro DNase I footprints of the 300 bp promoter using nuclear protein extracts from early and late blastulae are not detectably different and neither this sequence, nor a longer one extending to −1255, reproduces the loss of endogenous SpHE transcriptional activity after very early blastula stage. These observations imply that temporal repression of SpHE transcription involves a decrease in accessibility of the promoter to activators that are nevertheless present in nuclei and capable of activating transgene promoters. Temporal, but not spatial, downregulation is therefore likely to be regulated by negative activities functioning outside the −1255 promoter region which may serve as direct repressors or mediate an inactive chromatin structure.
Fang Liu - One of the best experts on this subject based on the ideXlab platform.
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a human mad protein acting as a bmp regulated transcriptional activator
Nature, 1996Co-Authors: Fang Liu, Akiko Hata, Julie C Baker, Jacqueline Doody, Juan M Carcamo, Richard M Harland, Joan MassagueAbstract:The TGF-beta/activin/BMP cytokine family signals through serine/threonine kinase receptors, but how the receptors transduce the signal is unknown. The Mad (Mothers against decapentaplegic) gene from Drosophila and the related Sma genes from Caenorhabditis elegans have been genetically implicated in signalling by members of the bone-morphogenetic-protein (BMP) subfamily. We have cloned Smad1, a human homologue of Mad and Sma. Microinjection of Smad1 messenger RNA into Xenopus Embryo Animal caps mimics the mesoderm-ventralizing effects of BMP4. Smad1 moves into the nucleus in response to BMP4. Smad1 has transcriptional activity when fused to a heterologous DNA-binding domain, and this activity is increased by BMP4 acting through BMP-receptor types I and II. The transactivating activity resides in the conserved carboxy-terminal domain of Smad1 and is disrupted by a nonsense mutation that corresponds to null mutations found in Mad and in the related gene DPC4, a candidate tumour-suppressor gene in human pancreatic cancer. Additionally, we show that DPC4 contains a transcriptional activation domain. The results suggests that the Smad proteins are a new class of transcription factors that mediate responses to the TGF-beta family.
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A human Mad protein acting as a BMP-regulated transcriptional activator
Nature, 1996Co-Authors: Fang Liu, Akiko Hata, Julie C Baker, Jacqueline Doody, Juan M Carcamo, Richard M Harland, Joan MassagueAbstract:THE TGF-β/activin/BMP cytokine family signals through serine/threonine kinase receptors, but how the receptors transduce the signal is unknown. The Mad (Mothers against decapentaplegic) gene from Drosophila ^1 and the related Sma genes from Caenorhabditis elegans ^2 have been genetically implicated in signalling by members of the bone-morphogenetic-protein (BMP) subfamily. We have cloned Smad1 , a human homologue of Mad and Sma . Microinjection of Smad1 messenger RNA into Xenopus Embryo Animal caps mimics the mesoderm-ventralizing effects of BMP4. Smad1 moves into the nucleus in response to BMP4. Smad1 has transcriptional activity when fused to a heterologous DNA-binding domain, and this activity is increased by BMP4 acting through BMP-receptor types I and II. The transactivating activity resides in the conserved carboxy-terminal domain of Smad1 and is disrupted by a nonsense mutation that corresponds to null mutations found in Mad and in the related gene DPC4 , a candidate tumour-suppressor gene in human pancreatic cancer^3. Additionally, we show that DPC4 contains a transcriptional activation domain. The results suggests that the Smad proteins are a new class of transcription factors that mediate responses to the TGF-β family.