Isolation And Function Research Of A Novel Cofactor In Estrogen Signaling

Estrogen receptor (ER) is a member of a large superfamily of nuclear receptors that regulate the transcription of estrogen-responsive genes and contains two subtypes: ERαand ERβ, which are ligand-dependent transcription factors. ER plays an important role in the development of breast cancer. Recently, endocrine therapy of breast cancer is acquired mainly by decreasing ER transcriptional activity. ER is thought to act as a target of the therapy of breast cancer and good index of prognosis.By far ,We are not very clear about the proteins interacting with different domains of ER and regulating its transcriptional activity , and even more ,how many proteins participate in regulating ER transcriptional activity when the level of estrogen decreases. Therefore, it is of great significance to identify and characterize the proteins regulating ER transcriptional activity in order to develop drugs for breast cancer. To under how ER functions ,with ERαAF1 as bait, Memo (mediator of ErbB2-driven cell motility) was identified by yeast two-hybrid.Since ERαcan bind to ERE(estrogen responsive element), ERE-containing luciferase reporter assay was used to determine the effects of Memo on the transcription activity of ERα. In this study, We found that Memo only enhanced ERα-dependent transcriptional activity in a ligand-independent manner, and Memo also enhanced ERαtranscriptional activity in a dose-dependent manner and mainly through ERαAF1 domain, We further used some other report genes regulated by estrogen, C3-luc and PS2-luc, and Memo also increases the transcriptional activity of these report genes.To further confirm the specific interaction between Memo and ERαboth in vitro and in vivo, we finished GST pull-down and co-immunoprecipitation experiments, and the results are consistent with the result of yeast two-hybrid screens. We found that Memo could interact with ERαboth in vitro and in vivo and specially bind to ERαAF1 in GST pull-down.Subsequently, the expression vectors of Memo siRNAs were constructed and confirmed by DNA sequencing. Western blot analysis showed that Memo siRNAs could effectively inhibit the expression of Memo gene. We also found that the ability of Memo to enhance ERE-luc reporter gene transcription was inhibited when MCF-7 cells were cotransfected with Memo siRNAs.To further understand the fuction of Memo, we made the polyclonal antibody of Memo by immuning Balb/c mouse. Western blot analysis indicated that the antibody is characteristic of high specificity and high titer, which is close to the level of anti-FLAG, then we determined the distribution of Memo in rat tissues using the high specific polyclonal anti-Memo antibody.Taken together, these results show Memo is a new regulating cofactor, which may plays an important role in estrogen signaling pathway through interaction with ERα. Further research will help us understand its specific regulating mechanism in estrogen signaling pathway. Therefore, Memo may represent a new target for therapeutic intervention in the future.