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Characterization of a novel factor that binds to the upstream element in the secretin gene enhancer to activate transcription
April 2000
AGAA86li
4809
4811
SUCRASE·ISOMALTASE GENE EXPRESSION REQUIRES HE· PATOCYTE NUCLEAR FACTOR·l DNA ELEMENT DURING MOUSE INTESTINAL DEVELOPMENT AND IS DlFFEREN· TIALLY MODULATED BY HNF-IA AND HNF·1B. Francois Boudreau, Peter G. Traber, Univ of Pennsylvania, Philadelphia, PA. The mouse sucrase-isomaltase (SI) gene is an enterocyte-specific gene expressed in a complex developmental pattern. We previously reported that a short, evolutionarily conserved gene promoter regulates developmental expression of SI in mouse small intestine. HNF-Ia and HNF-I{3 are transcription factors that interact with this short gene promoter through the SIF3 DNA element. We investigated the role of the SIP3 element and its cognate nuclear binding proteins to regulate SI gene expression. Transgenic mice were made using constructs harboring a two base mutation in the SIF3 element (SIP3m) that linked either nucleotides 201 to +54 or nucleotides -8500 to + 54 of the mSI gene to the human growth hormone (hGH) gene. Transgenic mice were generated with these constructs and the distribution and expression level of the transgene was evaluated at mRNA and protein levels. Transgenic lines derived from construct 201+54SIF3mmSIlhGH showed no detectable level of hGH expression as compared with the wild-type construct. Transgenic lines with the large mutated construct showed an overall 90% of reduction in hGH mRNA levels along the intestinal tract as compared to the wild-type construct. hGH protein was weakly detected in some regions of the proximal jejunum with a regional distribution similar to the wild-type promoter. To further evaluate the respective role of HNF-laand HNF-lj3in the regulation of SI gene, we used the Caco-2 intestinal cell line. Targeted diminution of endogenous HNF-l a protein levels with the use of antisense oligonucleotides resulted in a reduction of SI mRNA levels in post-confluent Caco-2 cells. Overexpression of HNF-l {3 protein in these cells led to a decrease of endogenous SI gene expression at late post-confluent stage. Taken together, these findings demonstrate that a single DNA element provides an important mechanism for modulating transcription of the SI gene during intestinal development and enterocyte differentiation.
CHARACTERIZATION OF A NOVEL FACTOR THAT BINDS TO THE UPSTREAM ELEMENT IN THE SECRETIN GENE ENHANCER TO ACTIVATE TRANSCRIPTION. Subir K. Ray, Andrew B. Leiter, New England Med Ctr, Boston, MA.
4810 BUTYRATE.DEPENDENT ACTIVATION OF P21 wAF1IS POTENTIATED BY ZBP·89. Longchuan Bai, Juanita L. Merchant, HHMIlUniversity of Michigan, Ann Arbor, MI. ZBP-89 is a Kruppel-type zinc finger transcription factor that binds to GC-rich elements in a variety of promoters including gastrin, ornithine decarboxylase, enolase, procollagen, stromelysin, vimentin and p21wAF1. ZBP-89 transcriptionally represses or activates depending on the target promoter. For example, ZBP·89 is a repressor on the gastrin and ODC promoters, but is an activator on the stromelysin promoter. ZBP-89 is differentially expressed in gastric cancers and its overexpression arrests pituitary cell lines in G1. AIM: The aim of this study was to anali;ze whether ZBP-89 mediates growth arrest thro~h activation of p21W Fl. gene expression coinciMETHODS: Since butyrate stimulates p2l w dent with growth arrest and differentiation in HT-29 cells, we studied the wAF1 regulation of the p21 promoter by ZBP-89 in these cells. RNase protection was used to quantify changes in ZBP-89 in RNA with butyrate treatment. The pCMVZBP-89 sense and antisense expression vectors were transiently co-transfected with the 2.3 p2l wAF1-Luc reporter prior to treatment with 5 mM butyrate. The site of ZBP-89 binding to the p21wAF1 promoter was identified by DNasel footprinting and EMSAs. Point mutations of the p21wAFI binding sites within the context of the 2.3 kb promoter was performed to identify the minimal binding site. RESULTS: Butyrate stimulated a 4-fold increase in ZBP-89 mRNA within 3 days. ZBP-89 co-transfection with the p2l wAFlreporter construct repressed slightly basal promoter activity. In the absence of ZBP-89, the p2l wAF1promoter was stimulated -6-fold with 5 mM bu~rate. However in the presence of ZBP-89 overexpression, the p2l wA 'promoter was stimulated 16-fold. Thus, the presence of ZBP-89 potentiated the activation of the p2l wAF1promoter by butyrate. DNasel footprinting analysis with recombinant ZBP-89 identified a ZBP-89 binding site at -245 to -216. EMSAs including competition with a series of mutant binding sites revealed that the core binding site for ZBP-89 was GGGAGG. We also found that ZBP-89 competes with Spl for this binding site. An antisense ZBP-89 expression vector abolished the potentiation as did point mutations of the core binding site. CONCLUSIONS: We conclude that ZBP-89 gene expression is activated by Na butyrate. ZBp·89 regulates the p21 promoter in a butyrate-dependent manner through a G-rich element. Since activation of the p2l promoter by butyrate correlates with growth arrest, ZBP-89 may retard cell growth through its ability to activate p21.
Cell type specific transcription of the secretin gene is regulated by a multielement enhancer between 174 and 53. Introduction of mutations between 174 to 160 reduces promoter activity by up to 80%. Our aim was to characterize the factors that bind to this upstream element. Using nuclear extracts from secretin producing HIT cells in mobility shift assays, we identified a DNA-protein complex that bound to this element but not to oligonucleotides containing mutations which reduced transcription. This complex is also cell specific as it was not seen in the same assay with nuclear extracts from the related cell line, INRIG9, that does not produce secretin. A multimeric copy of the binding site did not significantly increase the activity of a heterologous promoter, suggesting that the protein binding to this region may not directly function as transcriptional activator. Analysis of the proteins binding to the element with several antibodies revealed that antisera against the basic helix loop helix protein BETA2 supershifted bound factors, suggesting that BETA2 may form a complex with these proteins. BETA2 is a strong activator of secretin gene transcription that binds directly to a downstream E-box motif but not to the upstream element. To characterize the upstream element binding factors, we fractionated nuclear extracts on heparin-agarose followed by either a DE52-sepharose or a oligonucleotide affinity (binding site) column and assayed each fraction for DNA-binding activity. We observed a total loss of DNA-binding activity after the DE52 or affinity steps. Mixing experiments with the column fractions revealed that loss of binding activity resulted from physical separation of a nuclear protein Ref-l (redox factor-I) during fractionation. Consistent with this result, recombinant Ref-l restored lost binding activity. Ref-l stimulates DNA-binding activity of several transcription factors including API, Spl , NFKB, CREB, Egr 1 and p53 by reducing cysteine residues near the DNA binding domain. We screened a HIT cell cDNA expression library with a binding site probe in the presence of bacterially expressed Ref-l and isolated a partial cDNA encoding a new zinc finger protein. Our results suggest that a factor binding to the upstream element in the secretin enhancer increases transcription by a potentially novel mechanism. Rather than directly increase transcription, this factor may associate with other proteins that bind to the enhancer to further increase their transactivation. 4812 PHYSICAL INTERACTION BETWEEN THE KRUPPEL-LIKE TRANSCRIPTIONAL FACTORS ZF9IKLF6 AND GKLFIKLF4 AND ITS EFFECT ON THE HUMAN KERATIN 4 PROMOTER. Jun-ichi Okano, Oliver G. Opitz, Hiroshi Nakagawa, Timothy D. Jenkins, Scott L. Friedman, Anil K. Rustgi, Div of Gastroenterology, Univ of Pennsylvania, Philadelphia, PA; Div of Liver Desease, Mt Sinai Sch of Medicine, New York, NY. Introduction :Zf9 (CPBP/KLF6) is a widely expressed member of the Kriippel-Iike family of transcriptional factors which led us to evaluate its expression and function in the esophageal squamous epithelium, a model for the equilibrium between proliferation and differentiation. Methods :RT-PCR, Western blot, and immunohistochemical techniques were employed to examine Zf9 expression in the esophagus. Immunoprecipitation (lP) and Western blots were performed to examine a physical interaction between Zf9 and GKLF/KLF4, which is also a member of the Kriippel-like family expressed in the esophageal squamous epithelium. For functional analysis of Zf9 and GKLF on the differentiation-linked human keratin 4 (K4) promoter, transient transfections were performed in esophageal squamous cancer cell lines using a liposome mediated method. Results :RTPCR and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines and localized to the esophageal squamous epithelium by immunohistochemistry. Zf9 transactivated the human K4 promoter reporter gene construct in esophageal cancer cell lines. Co-transfection of Zf9 and GKLF led to coactivation of the human K4 promoter in an additive fashion. Further analysis by IP-Western revealed that there is a physical interaction between Zf9 and GKLF. Conclusions : We demonstrate a novel finding for Kriippel-like family members that Zf9 and GKLF can physically interact. This is important in regulation of human K4 gene transcription which is important in the switch from proliferation to differentiation in the esophageal epithelium.
AGAA86li
4809
4811
SUCRASE·ISOMALTASE GENE EXPRESSION REQUIRES HE· PATOCYTE NUCLEAR FACTOR·l DNA ELEMENT DURING MOUSE INTESTINAL DEVELOPMENT AND IS DlFFEREN· TIALLY MODULATED BY HNF-IA AND HNF·1B. Francois Boudreau, Peter G. Traber, Univ of Pennsylvania, Philadelphia, PA. The mouse sucrase-isomaltase (SI) gene is an enterocyte-specific gene expressed in a complex developmental pattern. We previously reported that a short, evolutionarily conserved gene promoter regulates developmental expression of SI in mouse small intestine. HNF-Ia and HNF-I{3 are transcription factors that interact with this short gene promoter through the SIF3 DNA element. We investigated the role of the SIP3 element and its cognate nuclear binding proteins to regulate SI gene expression. Transgenic mice were made using constructs harboring a two base mutation in the SIF3 element (SIP3m) that linked either nucleotides 201 to +54 or nucleotides -8500 to + 54 of the mSI gene to the human growth hormone (hGH) gene. Transgenic mice were generated with these constructs and the distribution and expression level of the transgene was evaluated at mRNA and protein levels. Transgenic lines derived from construct 201+54SIF3mmSIlhGH showed no detectable level of hGH expression as compared with the wild-type construct. Transgenic lines with the large mutated construct showed an overall 90% of reduction in hGH mRNA levels along the intestinal tract as compared to the wild-type construct. hGH protein was weakly detected in some regions of the proximal jejunum with a regional distribution similar to the wild-type promoter. To further evaluate the respective role of HNF-laand HNF-lj3in the regulation of SI gene, we used the Caco-2 intestinal cell line. Targeted diminution of endogenous HNF-l a protein levels with the use of antisense oligonucleotides resulted in a reduction of SI mRNA levels in post-confluent Caco-2 cells. Overexpression of HNF-l {3 protein in these cells led to a decrease of endogenous SI gene expression at late post-confluent stage. Taken together, these findings demonstrate that a single DNA element provides an important mechanism for modulating transcription of the SI gene during intestinal development and enterocyte differentiation.
CHARACTERIZATION OF A NOVEL FACTOR THAT BINDS TO THE UPSTREAM ELEMENT IN THE SECRETIN GENE ENHANCER TO ACTIVATE TRANSCRIPTION. Subir K. Ray, Andrew B. Leiter, New England Med Ctr, Boston, MA.
4810 BUTYRATE.DEPENDENT ACTIVATION OF P21 wAF1IS POTENTIATED BY ZBP·89. Longchuan Bai, Juanita L. Merchant, HHMIlUniversity of Michigan, Ann Arbor, MI. ZBP-89 is a Kruppel-type zinc finger transcription factor that binds to GC-rich elements in a variety of promoters including gastrin, ornithine decarboxylase, enolase, procollagen, stromelysin, vimentin and p21wAF1. ZBP-89 transcriptionally represses or activates depending on the target promoter. For example, ZBP·89 is a repressor on the gastrin and ODC promoters, but is an activator on the stromelysin promoter. ZBP-89 is differentially expressed in gastric cancers and its overexpression arrests pituitary cell lines in G1. AIM: The aim of this study was to anali;ze whether ZBP-89 mediates growth arrest thro~h activation of p21W Fl. gene expression coinciMETHODS: Since butyrate stimulates p2l w dent with growth arrest and differentiation in HT-29 cells, we studied the wAF1 regulation of the p21 promoter by ZBP-89 in these cells. RNase protection was used to quantify changes in ZBP-89 in RNA with butyrate treatment. The pCMVZBP-89 sense and antisense expression vectors were transiently co-transfected with the 2.3 p2l wAF1-Luc reporter prior to treatment with 5 mM butyrate. The site of ZBP-89 binding to the p21wAF1 promoter was identified by DNasel footprinting and EMSAs. Point mutations of the p21wAFI binding sites within the context of the 2.3 kb promoter was performed to identify the minimal binding site. RESULTS: Butyrate stimulated a 4-fold increase in ZBP-89 mRNA within 3 days. ZBP-89 co-transfection with the p2l wAFlreporter construct repressed slightly basal promoter activity. In the absence of ZBP-89, the p2l wAF1promoter was stimulated -6-fold with 5 mM bu~rate. However in the presence of ZBP-89 overexpression, the p2l wA 'promoter was stimulated 16-fold. Thus, the presence of ZBP-89 potentiated the activation of the p2l wAF1promoter by butyrate. DNasel footprinting analysis with recombinant ZBP-89 identified a ZBP-89 binding site at -245 to -216. EMSAs including competition with a series of mutant binding sites revealed that the core binding site for ZBP-89 was GGGAGG. We also found that ZBP-89 competes with Spl for this binding site. An antisense ZBP-89 expression vector abolished the potentiation as did point mutations of the core binding site. CONCLUSIONS: We conclude that ZBP-89 gene expression is activated by Na butyrate. ZBp·89 regulates the p21 promoter in a butyrate-dependent manner through a G-rich element. Since activation of the p2l promoter by butyrate correlates with growth arrest, ZBP-89 may retard cell growth through its ability to activate p21.
Cell type specific transcription of the secretin gene is regulated by a multielement enhancer between 174 and 53. Introduction of mutations between 174 to 160 reduces promoter activity by up to 80%. Our aim was to characterize the factors that bind to this upstream element. Using nuclear extracts from secretin producing HIT cells in mobility shift assays, we identified a DNA-protein complex that bound to this element but not to oligonucleotides containing mutations which reduced transcription. This complex is also cell specific as it was not seen in the same assay with nuclear extracts from the related cell line, INRIG9, that does not produce secretin. A multimeric copy of the binding site did not significantly increase the activity of a heterologous promoter, suggesting that the protein binding to this region may not directly function as transcriptional activator. Analysis of the proteins binding to the element with several antibodies revealed that antisera against the basic helix loop helix protein BETA2 supershifted bound factors, suggesting that BETA2 may form a complex with these proteins. BETA2 is a strong activator of secretin gene transcription that binds directly to a downstream E-box motif but not to the upstream element. To characterize the upstream element binding factors, we fractionated nuclear extracts on heparin-agarose followed by either a DE52-sepharose or a oligonucleotide affinity (binding site) column and assayed each fraction for DNA-binding activity. We observed a total loss of DNA-binding activity after the DE52 or affinity steps. Mixing experiments with the column fractions revealed that loss of binding activity resulted from physical separation of a nuclear protein Ref-l (redox factor-I) during fractionation. Consistent with this result, recombinant Ref-l restored lost binding activity. Ref-l stimulates DNA-binding activity of several transcription factors including API, Spl , NFKB, CREB, Egr 1 and p53 by reducing cysteine residues near the DNA binding domain. We screened a HIT cell cDNA expression library with a binding site probe in the presence of bacterially expressed Ref-l and isolated a partial cDNA encoding a new zinc finger protein. Our results suggest that a factor binding to the upstream element in the secretin enhancer increases transcription by a potentially novel mechanism. Rather than directly increase transcription, this factor may associate with other proteins that bind to the enhancer to further increase their transactivation. 4812 PHYSICAL INTERACTION BETWEEN THE KRUPPEL-LIKE TRANSCRIPTIONAL FACTORS ZF9IKLF6 AND GKLFIKLF4 AND ITS EFFECT ON THE HUMAN KERATIN 4 PROMOTER. Jun-ichi Okano, Oliver G. Opitz, Hiroshi Nakagawa, Timothy D. Jenkins, Scott L. Friedman, Anil K. Rustgi, Div of Gastroenterology, Univ of Pennsylvania, Philadelphia, PA; Div of Liver Desease, Mt Sinai Sch of Medicine, New York, NY. Introduction :Zf9 (CPBP/KLF6) is a widely expressed member of the Kriippel-Iike family of transcriptional factors which led us to evaluate its expression and function in the esophageal squamous epithelium, a model for the equilibrium between proliferation and differentiation. Methods :RT-PCR, Western blot, and immunohistochemical techniques were employed to examine Zf9 expression in the esophagus. Immunoprecipitation (lP) and Western blots were performed to examine a physical interaction between Zf9 and GKLF/KLF4, which is also a member of the Kriippel-like family expressed in the esophageal squamous epithelium. For functional analysis of Zf9 and GKLF on the differentiation-linked human keratin 4 (K4) promoter, transient transfections were performed in esophageal squamous cancer cell lines using a liposome mediated method. Results :RTPCR and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines and localized to the esophageal squamous epithelium by immunohistochemistry. Zf9 transactivated the human K4 promoter reporter gene construct in esophageal cancer cell lines. Co-transfection of Zf9 and GKLF led to coactivation of the human K4 promoter in an additive fashion. Further analysis by IP-Western revealed that there is a physical interaction between Zf9 and GKLF. Conclusions : We demonstrate a novel finding for Kriippel-like family members that Zf9 and GKLF can physically interact. This is important in regulation of human K4 gene transcription which is important in the switch from proliferation to differentiation in the esophageal epithelium.