ER alpha and AP-1 Interact In Vivo with a Specific Sequence of the F Promoter of the Human ER alpha Gene in Osteoblasts

Estrogen-responsive genes often have an estrogen response element (ERE) positioned next to activator protein-1 (AP-1) binding sites. Considering that the interaction between ERE and AP-1 elements has been described for the modulation of bone-specific genes, we investigated the estrogen responsiveness and the role of these cis-elements present in the F promoter of the human estrogen receptor alpha (ER ) gene. The F promoter, containing the sequence analyzed here, is one of the multiple promoters of the human ER gene and is the only active promoter in bone tissue. Through electrophoretic mobility shift (EMSA), chromatin immunoprecipitation (ChIP), and re-ChIP assays, we investigated the binding of ER and four members of the AP-1 family (c-Jun, c-fos, Fra-2, and ATF2) to a region located approximately 800 bp upstream of the transcriptional start site of exon F of the human ER gene in SaOS-2 osteoblast-like cells. Reporter gene assay experiments in combination with DNA binding assays demonstrated that F promoter activity is under the control of upstream cis-acting elements which are recognized by specific combinations of ER , c-Jun, c-fos, and ATF2 homo- and heterodimers. Moreover, ChIP and re-ChIP experiments showed that these nuclear factors bind the F promoter in vivo with a simultaneous occupancy stimulated by estrogen. Taken together, our findings support a model in which ER /AP-1 complexes modulate F promoter activity under conditions of estrogen stimulation.