ESTROGEN-RELATED RECEPTOR, HERR1, MODULATES ESTROGEN RECEPTOR-MEDIATED RESPONSE OF HUMAN LACTOFERRIN GENE PROMOTER

We have shown previously that estrogen-stimulated transcription from t he human lactoferrin gene in RL95-2 endometrium carcinoma cells is med iated through an imperfect estrogen response element (ERE) at the 5'-f lanking region of the gene. Upstream from the ERE, a DNA sequence (-41 8 to -378, FP1) was selectively protected from DNase I digestion by nu clear extracts from endometrial and mammary gland cell lines. In this report, using the electrophoresis mobility shift assay, site-directed mutagenesis, and DNA methylation interference analyses, we show that t hree different nuclear proteins bind to the FP1 region (C1, C2, and C3 sites). The nuclear receptor, COUP-TF, binds to the C2 site. Mutation s in the C1 binding region abolish C1 complex formation and reduce est rogen-dependent transcription from the lactoferrin ERE. When the imper fect ERE of the lactoferrin gene is converted to a perfect palindromic structure, the enhancing effect of the C1 binding element for estroge n responsiveness was abolished. me isolated a complementary DNA (cDNA) clone from an RL95-2 expression library that encodes the C1 site-bind ing protein. The encoded polypeptide maintains 99% amino acid identity with the previously described orphan nuclear receptor hERR1. A 2.2-ki lobase mRNA was detected in RL95-2 cells by the newly isolated cDNA bu t not by the first 180 base pair of the published hERR1 sequence. By W estern analysis, a major 42-kDa protein is detected in the RL95-2 nucl ear extract with antibody generated against GST-hERR1 fusion protein. Finally, we show that the hERR1 interacts with the human estrogen rece ptor through protein-protein contacts.