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.
Hirayama
K
K
Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Shimoji
M
M
Swick
L
L
Meyer
A
A
Kapatos
G
G
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D051698:Activating Transcription Factor 2; D017398:Alternative Splicing / Q000502:physiology; D001483:Base Sequence; D015306:Biogenic Monoamines / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D015152:Blotting, Northern; D023081:CCAAT-Binding Factor / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D018983:DNA Footprinting; D006136:GTP Cyclohydrolase / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D012260:Ribonucleases; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D015306:Biogenic Monoamines; D023081:CCAAT-Binding Factor; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D014157:Transcription Factors; C402244:nuclear factor Y; D001708:Biopterins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D012260:Ribonucleases; D006136:GTP Cyclohydrolase; C003402:sapropterin