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We have used site-directed mutagenesis and a homologous transient transfection system to investigate the role of the two imperfect estrogen response elements (EREs) located at -302/-334 in the 5'-flanking region of the estrogen-regulated Xenopus laevis vitellogenin B1 gene. Deletion of either ERE effectively abolishes estrogen-dependent transcription of the vitellogenin promoter. Neither replacement of the two imperfect EREs with a single consensus ERE at -334, nor insertion of one or two consensus EREs at -359, restores full estrogen responsiveness to the mutant promoter. In competition gel mobility shift assays using the DNA binding domain of the Xenopus estrogen receptor, the consensus ERE was a severalfold more effective competitor than the two imperfect B1 EREs. These data suggest that flanking DNA sequences may exert a significant effect on the activity of EREs as hormone-dependent transcription activators. When the imperfect EREs at -302/-334 were present, an additional consensus ERE at -359 exhibited synergistic activation of transcription. However, two consensus EREs located close to the TATA box showed additive, not synergistic, activation of transcription. In contrast, synergistic activation of transcription was observed in synthetic promoters containing two EREs and either the vitellogenin activator element or the NF1 or AP1 upstream activator elements.

The role of estrogen response elements in expression of the Xenopus laevis vitellogenin B1 gene.