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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

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.