Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene.

Abstract

Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobase (kb) of 5'-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active is estrogen receptor binding was identified within this new sequence, approximately 4 kb 5' of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.