A TGACG MOTIF MEDIATES GROWTH-HORMONE-FACTOR-1 PITUITARY-TRANSCRIPTIONAL-ACTIVATOR-1-DEPENDENT CAMP REGULATION OF THE RAINBOW-TROUT GROWTH-HORMONE PROMOTER/

The mechanisms involved in the regulation of the rainbow trout growth hormone (tGH) gene promoter by the pituitary-specific transcription fa ctor GHF1 (growth hormone factor 1), also called Pit1 (pituitary trans criptional activator 1), and cAMP have been investigated in mammalian and fish cells. The -340 to +24 5'-flanking region of the tGH gene fus ed to the luciferase gene was activated in rat pituitary GC cells and in HeLa cells cotransfected with an effector plasmid encoding rat GHF1 . GC cell nuclear extracts produced four GHF1-specific footprints (sit es F1 to F4) on the tGH promoter, each containing multiple W(4)NCAT (W , A or T) or closely related motifs. Mutational analysis performed in GC cells indicated that the proximal F1 site alone can direct transcri ption, but that the region encompassing the F2 and F3 sites is necessa ry for optimal activation and contains a TGACG motif (cAMP-response el ement, CRE) confering cAMP responsiveness. The role of the TGACG motif in mediating cAMP regulation of the tGH promoter was confirmed in pri mary cultures of trout pituitary cells. Cotransfection studies in carp EPC cells using an effector plasmid encoding trout GHF1 demonstrated the GHF1 dependence of a cAMP stimulation. Gel shift and southwestern experiments revealed nuclear proteins of 43 kDa and 30 kDa in GC and f ish cells, respectively, that bind specifically to the tGH CRE, sugges ting the involvement of CRE-binding protein/activating-transcription-f actor-1-related peptides in cAMP response. Incidentally, and in contra st with previous reports, we found the rat tGH promoter, that lacks TG ACG motifs, unresponsive to cAMP. Thus, the cAMP stimulation of the tG H gene is more similar to its human counterpart, that is also GHF1 dep endent and mediated by TGACG motifs in the promoter. It is suggested t hat control of GH gene expression has evolved modularly, through vario us assortments of the same regulatory units, rather than molecularly, through innovative units.