Estrogen receptor alpha (ERalpha) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERalpha binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERalpha has HRE DNA-binding activity, we evaluated the "EAAE" ERalpha, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERalpha null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERalpha tethering, because KIKO ERalpha effectively stimulates transcription using HRE motifs. The EAAE-ERalpha DNA-binding domain mutant mouse demonstrates that ERalpha DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERalpha tethering may not contribute to estrogen responsiveness in vivo.
Type of Publication:
Journal article published