FOXA1 promotes AR target gene expression by interaction with AR F

FOXA1 promotes AR target gene expression by interaction with AR FOXA1 can target a series new of transcription factors repre senting anywhere from several to hundreds of genes. To address whether the effects of FOXA1 on AR down stream targets are primarily through upregulating AR, rather than upregulating AR downstream targets dir ectly, we used untransfected MFE 296 cells and MFE 296 cells transfected with shFOXA1, NC, or shFOXA1 to gether with exAR. qRT PCR and western blotting analysis confirmed that transfection of exAR resulted in overexpression of AR. qRT PCR verified that MFE 296 shFOXA1 cells exhibited substantial decreases in the four AR tar gets compared with MFE 296 NC cells. Furthermore, cotransfection with exAR rescued the inhib ited expression of the target genes caused by FOXA1 downregulation in MFE 296 shFOXA1 cells.

In addition, we used untransfected AN3CA cells and AN3CA cells transfected with NC, ex FOXA1, or exFOXA1 together with siAR. qRT PCR and western blotting analysis confirmed that transfection with siAR re sulted in silencing of AR. Overexpression of FOXA1 increased the expression of the four AR target genes. Moreover, cotransfection with siAR partially re versed the FOXA1 induced overexpression. These results verified that AR downregulation attenuated the effect of FOXA1 on AR mediated transcription and suggested that FOXA1 might promote AR downstream targets at least in part through AR. FOXA1 and AR are found in the same protein complex To investigate whether FOXA1 affects AR mediated tran scription through binding to AR, we performed co immu noprecipitation experiments.

We used nuclear lysates from MFE 296 cells to conduct immunoprecipitation with anti FOXA1. FOXA1 co immunoprecipitated with AR, whereas immunoprecipitation with the isotype IgG con trol did not pull down AR or FOXA1, indi cating that FOXA1 interacted with AR in MFE 296 cells. We Volasertib supplier also performed the co immunoprecipitation experiment in AN3CA cells, which has low level of AR. As shown in Figure 4B, AR could be immunoprecipi tated by anti FOXA1 in the presence of DHT but not in its absence. This result indicated that FOXA1 and AR interacted physically. It is likely that FOXA1 affects AR mediated transcription via binding with AR. We further examined whether FOXA1 and AR could bind to the five putative FOXA1 AR binding regions, including the promoter and enhancer regions upstream of the TSS of AR target genes such as MYC. Our ChIP assays showed that both FOXA1 and AR could bind to all the five putative FOXA1 AR binding regions in MFE 296 cells. More over, both FOXA1 and AR bound most greatly to the Enh 1 region among the five binding regions.

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