Transcriptional regulation by estrogen of episomal prolactin gene regulatory elements

As a first step in defining the role of chromatin structure in steroid-regulated gene transcription, we have established a steroid-responsive minichromosome system that contains the 5' upstream regulatory region of the rat PRL gene (PRL) from -10 to -1960-basepairs fused to the antibiotic resistance gene, Tn5. The hybrid gene was inserted into a bovine papilloma virus (BPV) vector and then transfected into GH3 cells. Southern analysis of total genomic DNA revealed that the PRL-Tn5-BPV DNA existed in the cells as unrearranged episomes or minichromosomes at a level of 25-100 copies/cell. We monitored the estrogen responsiveness of the minichromosome-based PRL regulatory regions by measuring Tn5 mRNA levels. Treatment of GH3 cells for 48 h with 10 nM 17 beta-estradiol (E2) increased Tn5 mRNA levels 3- to 6-fold over those in untreated cells. Concurrently, endogenous PRL mRNA levels were induced 8- to 15-fold. Using nuclear run-on assays, it was found that E2 increased PRL-Tn5 transcription rates approximately 3-fold over those in untreated cells. The induced transcription was mediated through the PRL elements and not through any other cis-acting elements within the minichromosome. The PRL elements that contain a functional enhancer are located 3' downstream of the BPV early gene promoters in the minichromosome. However, there was no detectable effect of the PRL enhancer on BPV early gene transcription. Thus, we have established a minichromosome system containing the transcriptional regulatory elements of the rat PRL gene that responds to E2 in a manner very similar to the endogenous rat PRL gene.