Transcription from the P2 promoter of the growth hormone receptor gene involves members of the Sp transcription factor family

Abstract

The P2 promoter of the gene for growth hormone receptor is developmentally regulated and is differentially active in a number of tissues. Little is known about the identity of the transcription factors that participate to effect this pattern of transcription. Deletion analysis and transient transfection were used to localize a previously identified cis-acting element within the sheep P2 promoter to between positions -99 and -87. Gel mobility-shift assays with nuclear extracts from Chinese hamster ovary (CHO-K1) fibroblasts revealed that this sequence encompasses an atypical binding site for both Sp1 and two isoforms of Sp3. A gel mobility-shift scan of promoter sequences between -88 and +21 indicated the existence of three other binding sites for Sp1 and Sp3. One of these, designated site II and found by using a probe spanning -74 to -54, corresponds to a classical GC box consensus sequence. Site III (-63 to -41) and site IV (-27 to -5) harbour atypical Sp1/Sp3-binding sequences. Site-directed mutagenesis of site II or site IV decreased promoter activity by approx. 40%, whereas a promoter construct incorporating both mutations exhibited negligible (approx. 1%) activity. Co-transfection of expression plasmids encoding either Sp1 or Sp3 significantly transactivated reporter gene activity from a P2 promoter construct carrying all four Sp1/Sp3-binding sites (8-fold compared with 7.1-fold induction respectively). Sp1 is known to interact with a variety of other transcription factors to regulate the transcription of a number of differentially expressed genes. The identification of four binding sites for Sp1 and Sp3 within the P2 promoter of the gene for growth hormone receptor might point to other factors that interact to regulate the activity of this promoter in different tissues during foetal and post-natal development.