Estrogen receptor-related receptor alpha 1 interacts with coactivator and constitutively activates the estrogen response elements of the human lactoferrin gene

The human estrogen receptor-related receptor (ERR alpha 1, NR3B1a) was show n to bind a steroidogenic factor binding element (SFRE), TCAAGGTCATC, 26 ba se pairs upstream from the estrogen response element (ERE) of the human lac toferrin gene promoter, A mutation made at SFRE significantly reduced estro gen-dependent transcription from the lactoferrin ERE in human endometrial c ells. In this study, we demonstrated that ERR alpha 1 binds both SFRE and E RE elements and constitutively transactivates the lactoferrin gene promoter . In DNase I footprinting protection analysis, both SFRE and ERE regions we re protected by glutathione S-transferase-ERR alpha 1 fusion protein. The r eceptor formed two protein-DNA complexes with either SFRE or ERE in electro phoresis mobility shift assay. Homodimerization of ERR alpha 1 was confirme d with the mammalian two-hybrid system. ERR alpha 1 activates reporter cons tructs containing various types of estrogen response elements in endometria l and non-endometrial cells in transient transfection experiments. Overexpr essing the coactivator, SRC1a or GRIP1, further enhances ERR alpha 1-induce d transcriptional activity. We demonstrated that the AF2 domain of ERR alph a 1 is essential for the transactivation function and that deletion or muta tion at this region abrogates the activation capability. Protein-protein in teraction between the SRC1a and ERR alpha 1 C terminus was confirmed with a GST glutathione S-transferase "pull-down" assay. When comparing ERR alpha 1 and the estrogen receptor alpha (ER alpha) in many of the experiments, we found that ER alpha can also bind SFRE of the lactoferrin gene and transac tivate the promoter activity in a ligand-dependent manner. The present stud y demonstrated that ERR alpha 1 binds similar DNA elements as ER alpha and confers its transactivation function constitutively. Therefore, ERR alpha 1 may actively modulate the estrogen response of lactoferrin gene as well as other estrogen-responsive genes.