Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN-BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter

Authors

  • Kei Hirayama,

    1. Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
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  • Mika Shimoji,

    1. Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
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  • Lance Swick,

    1. Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
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  • Amy Meyer,

    1. Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
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  • Gregory Kapatos

    1. Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
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Address correspondence and reprint requests to G. Kapatos, Department of Psychiatry and Behavioral Neurosciences, 2309 Scott Hall, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA. E-mail: gkapato@med.wayne.edu

Abstract

GTP cyclohydrolase I (GTPCH) gene expression was investigated in the human monoamine-containing neuroblastoma cell line SK-N-BE(2)M17. Northern blot analysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced forms of GTPCH mRNA were detected with this assay. Incubation with 8Br-cAMP, but not nerve growth factor or leukemia inhibitory factor, produced a rapid increase in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly between 10 and 24 h. Treatment with 8Br-cAMP did not significantly modify the stability of GTPCH mRNA but did increase GTPCH transcription as determined by transient transfection assays of a luciferase reporter construct containing 1171 bp of human GTPCH 5′-flanking sequence. Cis-acting elements required for maximal basal and cAMP-dependent transcription were localized by deletion analysis to the 146 bp proximal promoter. DNase I footprint analysis of the proximal promoter using SK-N-BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Sp1-like site and the other a combined CRE-Sp1-CCAAT-box element. EMSA and supershift assays demonstrated that the combined CRE-Sp1-CCAAT-box element recruits ATF-2 and NF-Y but not Sp1–4 or Egr-1–3. NF-Y binding was confirmed using pure recombinant human NF-Y protein. Transcription of the human GTPCH gene in human SK-N-BE(2)M17 cells is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF-Y and ATF-2.

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