Estrogen receptor-related receptor α1 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 shown to bind a steroidogenic factor binding element (SFRE), TCAAGGTCATC, 26 base pairs upstream from the estrogen response element (ERE) of the human lactoferrin gene promoter, A mutation made at SFRE significantly reduced estrogen-dependent transcription from the lactoferrin ERE in human endometrial cells. In this study, we demonstrated that ERR alpha 1 binds both SFRE and ERE elements and constitutively transactivates the lactoferrin gene promoter. In DNase I footprinting protection analysis, both SFRE and ERE regions were protected by glutathione S-transferase-ERR alpha 1 fusion protein. The receptor formed two protein-DNA complexes with either SFRE or ERE in electrophoresis mobility shift assay. Homodimerization of ERR alpha 1 was confirmed with the mammalian two-hybrid system. ERR alpha 1 activates reporter constructs containing various types of estrogen response elements in endometrial and non-endometrial cells in transient transfection experiments. Overexpressing the coactivator, SRC1a or GRIP1, further enhances ERR alpha 1-induced transcriptional activity. We demonstrated that the AF2 domain of ERR alpha 1 is essential for the transactivation function and that deletion or mutation at this region abrogates the activation capability. Protein-protein interaction 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 transactivate the promoter activity in a ligand-dependent manner. The present study 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.