We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitaryspecific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-l factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-l and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

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The human PRL gene is expressed outside pituitary lactotrophs in decidualized endometrium and lymphoid cells, but here the mRNA contains a 5'-untranslated region from an additional noncoding exon 1a. We have isolated a genomic DNA clone containing human PRL exon 1a with 2800 basepairs (bp) of 5'-flanking sequences. Sequencing locates exon 1a -5840 bp up-stream of the pituitary start site. To study its suspected regulatory function, various lengths of the 5'-flanking region were linked to the luciferase (Luc) reporter gene. Their ability to direct gene expression has been analyzed in transfection studies. The proximal 1620 bp of promoter sequence directed Luc expression in the T-lymphoid Jurkat cell line, and this was unaffected by 5'-deletion to the proximal 453 bp. However, further 5'-deletion to the most proximal 67 bp drastically reduced this activity by 90%. The exon 1a promoter was inactive in pituitary GH3 cells and HeLa cells; in contrast, the exon 1b pituitary promoter, active in GH3 cells, was inactive in Jurkat cells. DNase-I footprinting studies and further 5'- and 3'-deletion analysis identified factor-binding sites within an enhancer element located at -375/-212 bp, which contributed approximately 50% of the promoter activity.

This study examines the regulation of the human PRL (hPRL) gene promoter by intracellular calcium. Deletants of the 5'-flanking region of the hPRL gene and constructs consisting of the thymidine kinase promoter linked to the first or second proximal Pit-1 binding site were fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. With the complete 5-kilobase pair (kbp) hPRL promoter sequence the calcium channel agonist Bay K8644 induced a significant 2-fold increase in CAT reporter gene expression and the antagonist verapamil a 4.5-fold reduction, using GH3 cells cultured in physiological levels of calcium. The transcriptional response to calcium influx was similar with a series of 5'-deleted hPRL-CAT constructs including those that comprised the proximal (up to 740 bp) or distal (-1300- to -1700-bp) sequences alone. When treating cells cultured in low calcium conditions the induction with the hPRL promoter increased to 5-fold on the addition of exogenous calcium and Bay K8644. The pituitary-specific expression of the hPRL gene is conferred by the interaction of the pituitary-specific factor Pit-1 with several binding sites located in the 5'-flanking DNA, of which three are located in the proximal region. This suggested that Pit-1 binding sites may be involved in the calcium response.(ABSTRACT TRUNCATED AT 250 WORDS)