Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat GTP cyclohydrolase I gene

5812 base pairs of rat GTP cyclohydrolase I (GTPCH) 5'-flanking region were cloned and sequenced, and the transcription start site was determined for the gene in rat liver. Progressive deletion analysis using transient transf ection assays of luciferase reporter constructs defined the core promoter a s a highly conserved 142-base pair GC rich sequence upstream from the cap s ite. DNase I footprint analysis of this region revealed (5' -> 3') a Sp1/GC box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an E-box . Transcription from the core promoter in PC12 but not C6 or Rata cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis showed that bo th the CRE and CCAAT-box independently contribute to basal and cAMP-depende nt activity. The combined CRE and CCAAT-box cassette was also found to enha nce basal transcription and confer cAMP sensitivity on a heterologous minim al promoter. The addition of the Sp1/GC box sequence to this minimal promot er construct inhibited basal transcription without affecting the cAMP respo nse. EMSA showed that nuclear proteins from PC12 but not C6 or Rata cells b ind the CRE as a complex containing activating transcription factor (ATF)-4 and CCAAT enhancer binding protein beta, while both PC12 and C6 cell nucle ar extracts were recruited by the CCAAT-box as a complex containing nuclear factor Y, Overexpression of ATF-4 in PC12 cells was found to transactivate the GTPCH promoter response to cAMP. These studies suggest that the elemen ts required for cell type-specific cAMP-dependent enhancement of gene trans cription are located along the GTPCH core promoter and include the CRE and adjacent CCAAT-box and the proteins ATF-4, CCAAT enhancer-binding protein b eta, and nuclear factor Y.