Identification of the Estrogen Receptor Alpha Gene as a Potential Target of the Transcription Factor FoxL2.

Abstract

The estrogen receptor alpha (ERα) gene is known to be regulated by the forkhead transcription factors FoxM1 and FoxO3a in breast carcinoma cells. At least eight consensus forkhead response elements (FHREs) have been identified in the human ERα promoter. In this study, we aim to determine if the forkhead transcription factor FoxL2 functions to regulate the ERα gene. FoxL2 is critical to ovarian function during follicullogenesis and has also been shown to play an important role in sex determination. FoxL2 is expressed in granulosa cells during follicular development. Therefore, the KK1 mouse granulosa cell line was chosen as a model system for these studies. We have performed promoter analysis of a 1.5 kB sequence representing the mouse ERα gene promoter to identify putative FoxL2 binding sites (ACAACA). Two such sequences were identified 411 and 449 base pairs upstream of the TATA element. We have also used Real Time RT PCR to determine the effect of FoxL2 over-expression on ERα gene expression. To perform this study, KK1 cells were transiently transfected for 24 hours with either a FoxL2 expression vector or the empty vector pcDNA3.1 as a control. Cells were harvested and total RNA was purified. Real-Time RT PCR was performed using Applied Biosystems (AB) Taqman Assays on the AB 7200 System. AB RQ software was used to determine changes in levels of FoxL2 and ERα mRNA. Upon comparing levels of mRNA in cells over-expressing FoxL2 to the control cells expressing normal levels of FoxL2, we found that the level of FoxL2 mRNA increased upon introduction of the expression vector as expected. However, we were surprised to find that the level of ERα mRNA increased as a result of FoxL2 over-expression. These results suggest that FoxL2 is an activator of ERα gene expression in the KK1 granulosa cell line.