Mechanisms of estrogen receptor (ER) action in breast cancer cells: Role of ER AF-1 domain in ER/SP-1 transactivation and modulation of ER by the ring finger coactivator SNURF

17β-Estradiol (E2) induces expression of several genes via estrogen receptor (ER)-Sp1 protein interactions with GC-rich promoter elements in which Sp1 but not ER binds DNA. This study reports the ligand- and cell context-dependent ER_α/Sp1 and ER_β/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ER_α and ER_β proteins physically interact with Sp1, yet only ER _α/Sp1 responds to E2 and antiestrogens in breast, but not in endometrial cells. Studies of chimeric proteins in which the AF-1 domain of ER_α and ER_β have been exchanged indicate that ER/Sp1 transactivation occurs through the AF-1 domain of ER_α. In order to further characterize this observation, deletion constructs in the AF-1 domain of both ER_α and ER_α/β were made and transactivation studies indicated that the region between aa79–117 of this domain is important for activation at an Sp1 element.; SNURF is a small RING finger protein that binds the zinc finger region (ZFR) of steroid hormone receptors and enhances Sp1- and androgen receptor-mediated transcription in COS and CV-1 cells. Results presented in this dissertation demonstrate that SNURF coactivates ERα activation of both ERE and Sp1 promoters in both ZR-75 and MCF-7 breast cancer cells. In mammalian two-hybrid assays in ZR-75 cells, SNURF interactions were estrogen (E2)-dependent and were not observed with the antiestrogen ICI 182,780. Moreover, peptide fusion proteins that inhibit interactions between helix 12 of ERα with LXXLL box-containing proteins also blocked ERα coactivation by SNURF in an ERE promoter, but only minimally (<50%) reduced ERα coactivation of an Sp1 promoter. Although SNURF requires helix 12 of AF-2, prototypical AF-2 interacting proteins do not affect SNURF/ER/ERE, while TBP does cooperate to activate SNURF/ER/ERE. The results are consistent with a unique model for cooperative coactivation of ERα that requires ligand binding, repositioning of helix 12, recruitment of TBP, and interaction with SNURF which binds both ERα and TBP. Results from SNURF/ERα/Sp1, indicate that coactivation requires the DBD of ER, and mediates coactivation of both the AF-1 and AF-2 activities of ER. Analysis of SNURF mutants, indicate that SNURF must contact DNA, interact with Sp1, and interact with ER to properly coactivate ERα/Sp1.