Clustered point mutation analysis of the rat prolactin promoter

Abstract

To identify DNA regions important for basal and hormone-stimulated transcription of the rat PRL gene, a series of clustered point mutations were prepared within the immediate 5′ flanking region. DNA fragments representing the wild-type and 19 different linker-scanner mutations of the PRL gene were each linked to a luciferase marker gene, and the DNA constructs were transferred into GH3 pituitary tumor cells by electroporation. Luciferase activity was determined 24 h after transfection in extracts from control cells or cells treated with 0.5 mM chlorophenylthio-cAMP, 100 nM TRH, or 100 nM phorbol myristate acetate. The individual clustered point mutations covered a region from just up-stream of the TATA box (position -30) to a position 193 basepairs up-stream from the start of transcription. Five regions in which mutations produced substantial decreases in both basal and cAMP-, TRH-, or phorbol ester-stimulated expression of the marker gene were detected. Three of these regions (positions -41 to -58, -113 to -124, and -149 to -156) correspond to previously identified binding sites for the pituitary-specific, homeobox protein, Pit-1/GHF-1. The fourth and fifth regions do not correspond to Pit-1/GHF-1-binding sites and presumably represent sites for an unidentified factor. Within these regions, sequences with some similarity to a consensus cAMP response element and an AP-2-binding site have been detected. These data confirm the importance of Pit1/GHF-1 as a key factor in PRL gene transcription. In addition, the results suggest that additional transcription factors are probably required for efficient expression of the PRL gene. Several of the linker-scanner mutations appeared to reduce the ability of cAMP and phorbol esters to stimulate gene expression, including mutations in Pit-1-binding sites as well as the putative cAMP response element and AP-2-binding site. These findings suggest a role for Pit-1 as well as other transcription factors in mediating both basal and multihormone-stimulated PRL gene transcription.