Tissue specific trans-acting factor interaction with proximal rat prolactin gene promoter sequences.

Abstract

Using an exonuclease III protection assay, strong, reversible and tissue-specific binding of GH3 cell nuclear factors to proximal regions of the rat prolactin (rPrl) promoter (-31 to -77) has been detected. A second less prominent region of factor binding, that may have a correlate in HeLa cell extracts, was detected in the region (-155 to -180). The binding is eliminated in the presence of excess unlabelled rPrl promoter sequences (-423 to +38), excess unlabelled distal rPrl 5'-flanking sequences (-1960 to -1260) and SV40-enhancer/promoter sequences; it is largely unaffected by growth hormone (rGH) promoter and RSV-LTR sequences. A plasmid containing the proximal rPrl promoter sequences (-75 to +38) was also shown to be an avid inhibitor, at low concentrations, of rPrl promoter driven chloramphenicol acetyl transferase (CAT) gene expression in transient cotransfection competition studies; under these assay conditions distal rPrl 5'-flanking sequences and RSV and rGH promoter plasmids do not compete. The results emphasize the critical importance of proximal rPrl promoter sequences for prolactin gene expression in GH3 cells but recognize the related functional potential of more distal sequences.