Identification of Sp1 as the transcription factor for the alternative promoter P2 of the bovine growth hormone receptor gene

Growth hormone receptor (GHR) mRNA variants that differ in the 5'-untranslated regions (5'-UTR) have been isolated in various species. These 5'-UTR variants are generated from the use of alternative promoters and/or alternative splicing. The 5'-UTR 1B is one of the GHR 5'-UTR variants isolated in the bovine but its homologues are also present in other species. The 5'-UTR 1B is a predominant GHR 5'-UTR expressed in many tissues. In the present study, we screened a bovine genomic library and isolated a 1.7 kb bovine GHR genomic sequence including exon 1B and its 5' flanking region from which the GHR 5'-UTR 1B is generated. Using primer extension, two major transcription start sites were mapped in the bovine exon 1B. Transient transfection analysis of the 5' flanking region of exon 1B confirmed its promoter activity (termed P2) in both Hep G2 and BHK-21 cells. Furthermore, analysis of deletion promoter-reporter constructs found that the basal activity of P2 resided in the proximal region of P2. DNase I footprinting analysis and electromobility shift assay (EMSA) identified the ubiquitous transcription factor Sp1 as the binding protein to a GC box-containing DNA element within the proximal P2. Deletion of the GC box greatly reduced the activity of P2 in cell lines. The GC box-containing site also appeared to bind Sp1 in the nuclear extracts from diverse bovine tissues. This suggests that interactions of Sp1 with the GC box-containing element in the proximal region of P2 may be part of the mechanism for the expression of the bovine GHR 5'-UTR 1B in diverse tissues.