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Uterine Estrogen Receptor-DNA Complexes: Effects of Different ERE Sequences, Ligands, and Receptor Forms

1991; Oxford University Press; Volume: 5; Issue: 7 Linguagem: Inglês

10.1210/mend-5-7-959

1944-9917

S W Curtis, Kenneth S. Korach,

Steroid Chemistry and Biochemistry

Previous studies used the gel retardation assay to examine the binding of the mouse estrogen receptor (ER) to the estrogen-responsive element (ERE) from the vitellogenin A2 gene (VitA2ERE). Multiple specific complexes were formed when the ER was bound to various estrogen agonists or antagonists, or in the absence of bound hormone. The ERE from the human PS2 gene, which varies from the consensus ERE by one base change in the right arm, was used in this study to determine the effect of DNA sequence on ER-ERE interaction with various ligandreceptor complexes. Partially purified ligand-free soluble ER showed a 3-fold lower affinity for the PS2ERE than for the VitA2ERE, suggesting a possible influence of the imperfect DNA sequence on certain binding interactions. However, multiple complexes of similar affinity were formed with the PS2 sequence by nuclear ER regardless of the agonist or antagonist bound. In gel retardation experiments, antagonist (LY117018) nuclear ER complexes bound to either PS2 or VitA2ERE migrated more slowly than agonist complexes, indicating that the slower migrating form of the complex was not due to the DNA sequence. Interestingly, soluble ER bound by LY 117018 did not produce this decreased mobility complex, suggesting that it was specific to the nuclear form of the ER antagonist complex. Receptor activation has been linked with exposure to increased temperature, resulting in an ER form that has an increased affinity for DNA. The binding of molybdate-stabilized nonactivated 8S ER to VitA2ERE was studied to determine the effect of temperature on ER binding. Heat pretreatment of the stabilized soluble 8S ER did not cause an increase in ER-ERE binding during a 45-min reaction. Heat pretreatment slightly increased the final level of complex formed when followed for 24 h, but this temperature activation of the ER is also simply a reflection of an increased reaction rate of the complex formation between the ER and the ERE. Molybdate-stabilized soluble ER formed two specific ERE complexes. Partial purification of the stabilized soluble ER resulted in formation of only one specific ERE complex. This differential multiplicity of ER-ERE complexes formed with native ER extracts may be due to the presence of additional factors which contribute to the formation of the complexes. Studies are presently underway to identify these factors from uterine tissue. This study, using various uterine ER forms and different ERE sequences, suggests that the effect of estrogen on gene expression is not mediated only at the level of R-ERE interaction, but may occur through regulation of ER interaction with additional nuclear structures or proteins involved in transcription.