Identifying FoxA1 Interacting Partners: Insight into Androgen Regulated Prostatic Genes

Abstract

This research project is to identify FoxA1 interacting partners. In this study, a potential FoxA1 binding partner, Upstream Stimulatory Factor 2 (USF2), has been examined; its DNA binding activity was determined and the function of USF2 was monitored in USF2 knockdown LNCaP cells. FLAG-6His-FoxA1 stable expressing LNCaP cell lines were established. The FoxA1 protein complex from LNCaP tagged-FoxA1 cells was purified and analyzed by Liquid Chromatography Tandem Mass Spectrometry. USF2 has been confirmed to interact with FoxA1 using ImmunoPrecipitation and glutathione-S-transferase pull-down assays. Our study also indicates, as FoxA1, USF2 binds to prostatic specific gene promoters such as probasin promoter, Spermine Binding Protein promoter, and the Prostate Specific Antigen core enhancer. PSA mRNA levels were increased in USF2 knocked down LNCaP cells compared with control LNCaP cells when the LNCaP cells were treated with DHT (10-8 M). Using LC-MS/MS analysis, a total of 16 proteins that appear to interact with Foxa1 have been identified and eight of them have been previously reported as AR interacting proteins. Further work is needed to confirm the binding between FoxA1 and these identified proteins and to perform functional studies of the confirmed FoxA1 binding partners. This study has identified FoxA1 interacting partners which helps unravel the critical transcription factors that control cell type specific gene expression and to further understand the role of FoxA1 during prostate development. This work gives us an insight into the essential mechanism of androgen regulation of the prostate gland and of hormonal regulation of prostate cancer.