Molecular cloning, genomic organization, and identification of the promoter for the human pituitary tumor transforming gene (PTTG).

Recently, we cloned and sequenced cDNA of a potent pituitary tumor transforming gene (PTTG) from human testis and showed that this gene is expressed highly in various human tumors, including tumors of the pituitary and adrenal glands, and the liver, kidney, endometrium, uterus, and ovary. To determine the genomic organization of the PTTG and its transcriptional regulation in tumors, we isolated the gene. The PTTG spans more than 10kb and contains five exons and four introns. Primer extension and RNA protection assays indicated a transcription start site at an adenine residue at 37 bases upstream of the translation start site (ATG). Analysis of the 5' flanking region of the gene revealed the existence of three SP1/GC boxes, three AP1 and one AP2 binding sequences, a cyclic AMP response element sequence, and an insulin response element sequence. The promoter activity of the PTTG was evaluated by transfecting a human ovarian tumor cell line (SKOV3) and a mouse fibroblast cell line (NIH 3T3) with a chimeric fusion construct containing the 5' flanking sequence (nucleotide from -1336 to +34) and luciferase reporter gene (pluc 1370). The promoter activity of this construct was 210-fold higher in SKOV3 and 20-fold higher in NIH 3T3 cells than the promoterless vector. Deletion of sequences at the 5' end of the pluc 1370 construct from nucleotide -1336 to -1157 (pluc 1190), from nucleotide -1336 to -977 (pluc 1010) and from nucleotide -1336 to -707 (pluc 740) further increased luciferase activity. Further deletion of the 5' sequence from nucleotide -1336 to -407 (pluc 440), and from nucleotide -1336 to -127 (pluc 160) decreased activity by 95%. These results suggest that the sequence from nucleotide -126 to +34 is sufficient for PTTG promoter activity and that the sequence between nucleotide -706 and -407 contains an enhancer element. PTTG promoter activity was eight- to ten-fold higher in SKOV3 cells than NIH 3T3 cells, suggesting a basis for the tumor-specific expression of the PTTG. Knowledge of the genomic organization and the promoter region of the human tumor transforming gene will allow further studies of possible disorders of the PTTG as well as facilitate elucidation of the transcriptional control of PTTG expression in human tumors.