IDENTIFICATION OF THE SEQUENCES WITHIN THE HUMAN-COMPLEMENT-3 PROMOTER REQUIRED FOR ESTROGEN RESPONSIVENESS PROVIDES INSIGHT INTO THE MECHANISM OF TAMOXIFEN MIXED AGONIST ACTIVITY

The promoter of the human C3 gene has been shown to be responsive to s timulation by both estrogen and tamoxifen-activated estrogen receptor (ER) in transcriptional assays reconstituted in mammalian cells. Using a series of deletions and point mutations, we have determined that th e agonist activity of these two compounds was dependent upon the direc t interaction of ER with each of three estrogen response elements (ERE s) contained within this promoter. One of these sequences, ERE1 resemb les the canonical vitellogenin A2-ERE whereas the other two, EREP and ERE3, do not display significant homology to known EREs. Using gene tr ansfer studies it was shown that these sequences are necessary and suf ficient for ER-mediated transcription. Interestingly, using in vitro r eceptor/DNA-binding assays we demonstrated that neither ERE1, ERE2, or ERE3 alone formed high-affinity complexes with purified ER; however w hen a promoter fragment containing all three sequences was used, speci fic, high-affinity ER-DNA interactions were observed. It was not surpr ising, therefore, that, when assayed individually on a heterologous pr omoter, these sequences function as weak EREs but together they act in a synergistic manner to create a strong ER-dependent enhancer. It has been suggested that tamoxifen mediates its partial agonist activity t hrough AP-l at target promoters. However, the fact that purified ER ca n bind directly to the estrogen-responsive sequences within the C3 pro moter, and that tamoxifen activity on this promoter is unaffected by A P-1 coexpression, indicates that at least on some promoters tamoxifen can manifest partial agonist activity through a classical ER/ERE- medi ated mechanism.