Direct cooperation between androgen receptor and E2F1 reveals a common regulation mechanism for androgen-responsive genes in prostate cells

Abstract

We have studied the regulation of ATAD2 gene expression by androgens in prostate cells. ATAD2 is a coactivator of the androgen receptor (AR) and the MYC protein. We showed that ATAD2 expression is directly regulated by AR via an AR binding sequence (ARBS) located in the distal enhancer of its regulatory region. The gene is also regulated by the E2F1 transcription factor. Using knockdown and chromatin immunoprecipitation technique approaches, we could demonstrate that AR and E2F1 functionally collaborate and physically interact between each other. From the analysis of chromatin conformation, we conclude that this cooperation results from a chromatin looping over the ATAD2 promoter region between the ARBS and E2F1 binding site in an androgen-dependent manner. Furthermore, we could show that several genes overexpressed in prostate cancer and potentially involved in several aspects of tumor development have an ARBS and an E2F1 binding site in their regulatory regions and exhibit the same mechanism of regulation by both transcription factors as ATAD2.

Fig. 1.

Androgen-dependent ATAD2 expression. Expression of ATAD2 mRNA. Total RNA was isolated from control and R1881-treated LNCaP cells, and the mRNA levels of ATAD2 (A) and GAPDH (B) were quantified by RT-qPCR. Data represent means ± sd, n = 3. C, Binding assay for the AR-DBD protein on the ARBS of the ATAD2 promoter in vitro. EMSA were carried out on a ATAD2 promoter oligonucleotide containing the ARBS (left panel) or the nonspecific mutant oligonucleotide (right panel). D, DNase I footprinting of the binding of AR-DBD on ATAD2-ARBS. Sp.oligo and N-Sp.oligo refer, respectively, to WT and mutant ATAD2-ARBS. GL− indicates a guanine ladder for the corresponding oligonucleotide-labeled strand obtained after UV-laser irradiation and tormamidopyrimidine [fapy]-DNA glycosylase (FPG) treatment. Base sequences of the specific WT and mutant oligonucleotides are shown. The position of the AR-DBD footprint is denoted by vertical lines.

Fig. 2.

Study of the enhancer promoter. ChIP were performed on LNCaP cells treated with R1881 or untreated after cross-linking with formaldehyde. A, Schematic representation of the ATAD2 promoter. A2 and E2 represent the respective specific binding sites of AR and E2F. E1, E3, A1, and A3 are regions located 2 kb upstream and downstream of the specific binding sites of E2F and AR, respectively, and were used as controls for the binding specificity. B and C, ChIP assays on LNCaP cells using an anti-AR antibody and detection of binding on sites on the distal enhancer (B) and proximal (C) promoter regions. Values are the means of three independent experiments. Error bars indicate sd.

Fig. 3.

Cooperation between E2F1 and AR. A, Coimmunoprecipitation was realized on LNCaP cells with AR antibodies (upper panel) or with E2F1 antibodies (lower panel). B and C, Re-ChIP were performed on exponentially growing LnCaP cells 28 after cross-linking. Chromatin was precipitated first with an anti-AR antibody and then with an anti-E2F1 antibody. Control experiments were performed using IgG in place of the respective anti-AR and anti-E2F1 antibodies (B). D and E, E2F1 is essential for AR recruitment on the proximal promoter. ChIP experiment on LNCaP cells transfected with scrambled siRNA, siE2F1, or siAR. qPCR was carried out on the distal enhancer (D) and the proximal promoter regions of ATAD2 (E). F, Design of the 3C experiment with the localization of the BglII sites on the ATAD2 promoter and the primers used for the experiment. H, Loop between distal enhancer and proximal promoter of ATAD2. 3C was performed in LNCaP cells. Cells were transfected either with scrambled siRNA, siE2F1, or siAR and treated with R1881 or ethanol as vehicle. Results are fold enrichment on noncross-linked cells. Error bars indicate sd.

Fig. 4.

A, Venn diagram comparing the GEO datasets. AR ChIP-seq data (GSM686926) and E2F1 ChIP-seq data (GSM558469) were compared in order to identify genes the expression of which might be regulated by AR and E2F1. The result of this analysis was compared with microarray data performed on an adenocarcinoma specimen (GSD2171) to focus on genes expressed in prostate cancer. B, Expression profile of genes identified using the analysis described in A in LNCaP cells transfected with scrambled siRNA, siAR, or siE2F1. Results show the ratio between R1881-treated and nontreated cells. Results are means ± sd. C, Re-ChIP on LNCaP cells treated or not with R1881 as explained in Fig. 3B. qPCR was carried out on the ARBS and E2FBS of each gene. Results are fold enrichment on control IgG.