Genomic organization and identification of an enhancer element containing binding sites for multiple proteins in rat pituitary tumor-transforming gene

Abstract

The rat pituitary tumor transforming gene (PTTG) genomic structure was characterized in this study. Northern blot analysis showed that PTTG mRNA is highly expressed in testicular cell lines. Transfection of testicular cell lines with fusion constructs containing various portions of PTTG 5'-flanking sequences linked to luciferase showed that at least 745 base-pair (bp(s)) 5'-flanking sequences are required for PTTG transcriptional activation. DNaseI footprinting assays indicated that nuclear protein(s) from testicular cell lines interacts with PTTG 5'-flanking sequence between -509 and -624 bp, including two consensus Sp1 binding sites. Western and Southwestern blot analysis showed that three nuclear proteins in addition to Sp1 protein specifically interact with this DNA sequence and that two of these proteins are testicular cell-specific. Deletion of this 115-bp sequence from PTTG promoter resulted in complete loss of promoter function. Site-directed mutagenesis within the Sp1 consensus sequences indicated that the Sp1 binding sites are not critical components of the enhancer sequence for PTTG trancriptional activation in testicular cell lines. Finally, the 115-bp enhancer sequence was shown to be able to activate transcription from a heterologous promoter. These results suggest that PTTG transcriptional activation in testicular cell lines involves interactions of multiple nuclear factors with the PTTG 5' enhancer sequence.