The Role of DNA Binding in Transcription Activation by Estrogen Receptor

123 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.During the course of this study we created a superactive estrogen receptor by inserting of two copies of a well defined, synthetic 18 amino acid amphipathic helix into the hinge region of the Xenopus estrogen receptor (XER). The resulting mutant, XER/2AH, provides us with a useful tool to study the mechanisms of gene activation by estrogen. XER/2AH exhibited 2-3 fold increased hormone-dependent transcription. Using a minimal promoter containing two copies of the estrogen response element (ERE) linked to a TATA box, it exhibited a 10 fold increase in hormone-independent constitutive transcription activity compared to that of the wild-type XER. The hormone binding properties and protein expression levels of both the wild-type XER and the XER/2AH mutant are similar. Surprisingly, unliganded XER/2AH exhibited a striking 15-20 fold increase in DNA binding affinity over the wild-type XER. We also obtained the first evidence in vertebrates that binding to EREs by estrogen receptor is estrogen-dependent.In this study, we also addressed the potential role of mutant forms of estrogen receptor in chemotherapy, such as tamoxifen resistance during breast cancer treatment. We found that in certain types of cells, in the presence of tamoxifen, XER/2AH behaved almost exactly like wild-type XER in the presence of estrogen. Our data also provide strong evidence demonstrating that the antiestrogen compound ICI 164,384 is not a pure antagonist.Using deletion analysis we observed that the N-terminal and C-terminal transactivation domains play different roles in both DNA binding and cooperation with other transcription factors. We found that in a homologous system both of the transactivation domains were essential for fully active estrogen receptor. Moreover, we found that a repressor function for DNA binding was located in the N-terminal region of the receptor. Deletion of this region sharply increased DNA binding. (Abstract shortened by UMI.)U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD