REGULATION OF GONADOTROPIN-RELEASING-HORMONE TRANSCRIPTION BY PROTEIN-KINASE-C IS MEDIATED BY EVOLUTIONARILY CONSERVED PROMOTER-PROXIMAL ELEMENTS

We previously demonstrated that down-regulation of protein kinase C (PKC) by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment leads to the specific repression of GnRH transcription in GT1-7 hypothalamic neurons. Here we have investigated the regulatory sequences and cognate DNA-binding proteins that mediate this transcriptional response. The promoter-proximal section of the GnRH gene contains an evolutionarily conserved sequence that is bound along its entire length by GT1-7 nuclear proteins in DNase I protection assays. Two distinct regions within this sequence are required for PKC regulation of the GnRH gene, as excision of either region results in loss of TPA repression of transcription. Excision of either of these regions also decreases basal transcription, demonstrating their role in GnRH promoter function. One region encompasses three AT-rich protein-binding sites; the other is an extended region of continuous DNase I protection, 50 nucleotides in length, that contains consensus recognition motifs for the CCAAT/EBP and helix-loop-helix families of transcription factors. Mobility shift analysis of binding to the latter region reveals that TPA treatment of GT1-7 neurons induces the formation of a specific DNA-protein complex with kinetics of appearance consistent with a role in repression of GnRH transcription, Thus, the sequences that mediate PKC regulation of GnRH are proximal to the promoter, evolutionarily conserved, and form TPA-inducible complexes with GT1-7 nuclear proteins.