Figure 3. ERG and AR co-occupancy of target genes in prostate cancer.
(A) Overlap between the binding sites of different regulators. Significance of overlap was measured relative to their respective overlap with the NRSF binding sites. *P<0.05, ** P<0.01, and ***P<0.001 by hypergeometric test.
(B) ERG binds to a majority of the AR-bound genes. AR or ERG ChIP-Seq binding sites were each assigned to the nearest gene on the genome to derive the list of bound genes.
(C) Overlap between the ERG-bound genes and the genes that are bound by both AR and ERG as determined by re-ChIP-Seq using an anti-ERG and an anti-AR antibody.
(D) Representative genes co-occupied by AR and ERG. On the Y-axes are the number of reads of AR (left, in blue) and ERG (right, in red) binding sites. Above the plot are the TSS and direction denoted by arrows, exons by black box, and introns by horizontal lines, respectively. The genomic coordinates are indicated below the plot.
(E) ChIP-PCR confirms AR and ERG co-occupancy on selected genes. The Y-axis on the left represents AR ChIP enrichment in VCaP cells treated with R1881 normalized to the ethanol (Ethl) treated cells. The Y-axis on the right represents ChIP enrichment (in log scale) by an anti-ERG antibody normalized to IgG. Error bars: n=3, mean ± SEM. The 3′ intronic region of the KIAA0066 gene was used as a negative control.
(F) AR and ERG co-occupancy in a prostate cancer tissue. A prostate tumor tissue that expresses both AR and TMPRSS2-ERG were analyzed by ChIP-Seq.
(G) Physical interaction between the AR and ERG proteins. VCaP cells were immunoprecipitated by various antibodies and immunoblotted for AR. To deplete DNA, VCaP cell lysates were pre-incubated with ethidium bromide for 30 min before immunoprecipitation. Representative experiment of 4 independent co-immunoprecipitation assessments is shown.
(H) Interaction of ERG with AR in vitro via the ETS domain. HaloTag-ERG protein and mutants were generated by in vitro transcription/translation in wheat germ extracts. Recombinant GST-AR was incubated with the in vitro translated protein products and glutathione beads used in pull down assays. The interaction between AR and various domains of ERG are summarized and indicated as + or -. See also Figure S3 and Table S5.