New androgen receptor genomic targets show an interaction with the ETS1 transcription factor

Abstract

The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15-base pair bipartite palindromic androgen-response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on-chip detection of genomic fragments. We identified 1,532 potential AR-binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR-binding sites showed that more than 50% of AR-binding sites did not contain the established 15 bp AR-binding element. Unbiased sequence analysis showed 6-bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

Figure 1

Androgen receptor-bound gene promoters identified by ChIP-chip. (A) AR ChIP-chip profiles of eight AR target gene promoters from vehicle (+EtOH)- and androgen (+R1881)-treated experiments. Genomic locations are indicated on the x axis and enrichment compared with total input DNA is shown on the y axis. (B) AR ChIP and qPCR analysis of 26 AR promoter targets in LNCaP cells. (C) AR ChIP and qPCR analysis of eight promoter targets in DUCaP cells. Relative enrichment by ChIP was quantified by using the standard curve method following 1 h with vehicle (EtOH) or androgen (R1881) relative to β-actin control and total genomic DNA input (see the supplementary information online for details). Values are the averages of replicate experiments. Enrichment scores (S) and Wilcoxon scores (W) from LNCaP AR ChIP-chip analysis are indicated next to corresponding genes. AR, androgen receptor; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with on-array detection; Chr, chromosome; EtOH, ethyl alcohol; KLK, human glandular kellikrein; qPCR, quantitative PCR.

Figure 2

Functional annotation of androgen receptor ChIP target genes. (A) AR ChIP-chip target gene-enriched Gene Ontology categories and gene expression changes identified by comparing results with publicly available expression array data. (B–E) Heat-maps showing changes in gene expression for individual clinical samples, taken from publicly available expression array data (Varambally et al, 2005), for sets of genes identified as androgen-regulated (B), involved in transcription (C) and development (D). (E) Gene expression heat-map from a separate clinical expression array study for AR target genes that were identified as androgen-regulated (Tomlins et al, 2007). Arrows in B and E indicate that the expression of the genes selected have been reported to be upregulated on androgen treatment in previous studies. AR, androgen receptor; BN, benign; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with on-array detection; EPI-BPH, benign prostatic hyperplasia; EPI-NORM, normal prostate epithelium; GO, Gene Ontology; KLK, human glandular kellikrein; MET, metastatic PrCa; PrCa, primary PrCa.

Figure 3

Sequence analysis of androgen receptor-bound promoter regions. (A) Sequence logo of the in vitro-derived 15-bp ARE (SELEX) and known genomic inverted repeat (IR) and direct repeat (DR) ARE sequences. (B) Venn diagram showing the occurrence of 15-bp AREs in AR ChIP-chip-enriched sequences. (C) Sequence logos of AR ‘half-site' motifs identified in AR-bound promoter sequences using Nested MICA. (D) Venn diagram of the co-occurrence of the 15-bp ARE and 6-bp AR ‘half-site' motif 2 matrices in AR-bound sequences. (E) Sequences used in oligonucleotide pull-down assays, corresponding to wild-type (WT) and scrambled (MUT) sequences from the KLK2 promoter 15-bp ARE and UNQ9419 promoter 6-bp AR ‘half-site'. (F) AR western blot of oligonucleotide pull-down fractions, as indicated. Low-molecular-weight region of a parallel Coomassie blue-stained gel shows equal loading of cell lysates. (G,H) UCSC Genome Browser tracks showing 15-bp ARE and 6-bp AR ‘half-site' occurrence in KLK2 and UNQ9419 promoters. (I,K) AR ChIP and qPCR for KLK2 (I) and UNQ9419 (K) promoters from vehicle (+Etok)-and androgen (R1881)-treated experiments. (J,L) Cells were maintained in ethanol (+E) or treated on a time course with androgen (+R; R1881) for 18 h. Material was harvested at 1, 4 and 18 h during the time course and subjected to qrtPCR for KLK (J) and UNQ9419(L), respectively. AR, androgen receptor; ARE, androgen-response element; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with on-array detection; Chr, chromosome; KLK2, human glandular kellikrein; qrtPCR, quantitative real-time PCR.

Figure 4

ETS1 and androgen receptor interaction. (A) Sequence logos for ETS1 and AR ChIP-chip-enriched motifs 9 and 1. (B) UCSC Genome Browser tracks showing 15-bp ARE, 6-bp AR ‘half-site' and ETS1 motif occurrence in UNQ9419, CCNG2 and PRAME promoters. Locations of AR ChIP-chip peaks are indicated; darker boxes represent higher signal. (C) ETS1 ChIP and qPCR for UNQ9419, CCNG2 and PRAME promoters from vehicle (+EtOH)- and androgen (R1881)-treated experiments. (D,E) Quantitative real-time PCR of ETS1, UNQ9419, CCNG2 and PRAME 48 h after transfection with an ETS1 RNAi construct (pSR-ETS1) or a scrambled control (pS-Scr) (D) and ETS1 expression construct (+ETS1) or an empty vector control (pSG5) (E). qPCR data were quantified using the standard curve method, values relative to control sample and averages of replicate experiments. (F,G) Confocal microscopy images of LNCaP cells treated with androgen (+R1881) or vehicle (+EtOH) for 1 h. Cells were transfected with either AR–GFP expression construct (F) or pSG5 empty vector (G) and stained for AR (N20) and ETS1 (C20 and 1G11), as indicated. AR, androgen receptor; ARE, androgen-receptor elements; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with on-assay detection; DAPI, 4′, 6′-diamidino-2-phenylindole; ETS, avian erythroblastosis virus E26 homologue; GFP, green fluorescent protein; qPCR, quantitative PCR; RNAi, RNA interference.