DNA-BINDING OF HYPOTHALAMIC NUCLEAR PROTEINS ON ESTROGEN RESPONSE ELEMENT AND PREPROENKEPHALIN PROMOTER - MODIFICATION BY ESTROGEN

Preproenkephalin (PPE) gene expression is specifically induced by estr ogen in hypothalamus of ovariectomized (OVX) females, better than in m ale rats. To study estrogen actions on gene regulation, we have presen tly characterized protein-DNA interactions by use of a consensus estro gen response element (ERE) and a putative ERE from PPE gene, with nucl ear extracts from hypothalamus. By use of the electrophoretic mobility shift assay (EMSA), ERE binding activity was detected in nuclear extr acts from neuronal tissues including hypothalamus, hippocampus, striat um, cerebellum and frontal cortex, and non-neuronal tissues such as pi tuitary and uterus, but not lung of OVX female rats with a consensus E RE, as well as a 129-bp PCR fragment from PPE promoter and a hairpin o ligonucleotide that contains a putative ERE of the rat PPE gene. The E RE binding was eliminated by the addition of specific ERE-containing o ligonucleotide, but not control oligonucleotides. Protein and DNA asso ciated and dissociated very rapidly. By use of supershift assay, inter actions of estrogen receptor with ERE were demonstrated in hypothalami c nuclear extracts. The initial levels of specific ERE binding in the hypothalamic nuclear extracts were comparable between castrated male a nd OVX female rats. However, estrogen treatment, either estradiol or e stradiol benzoate, produced a rapid and tissue-specific induction of a slow mobility complex of ERE binding in hypothalamic nuclear extracts from females, better than in male rats, presumably from other associa ted factors, or a conformational change or other posttranslational mod ifications, This estrogen-induced slow mobility complex of ERE binding in hypothalamus was not observed after treatment with progesterone or tamoxifen. These results suggest that specific ERE binding is present in rat hypothalamic nuclear proteins, which may contribute to the upr egulation of PPE gene expression by estrogen, and that the sexually di fferentiated action of estrogen may be related to an estrogen-induced conformational change, but not to the initial level of ERE-binding act ivity.