(A) Correlation matrices for chromosome 6 obtained from RWPE1-ERG cells (Left) and RWPE1-GFP cells (Center) and the difference between the data from two cell lines [(RWPE1_ERG) − (RWPE1_GFP)] (Right). A schematic of chromosome 6 is given (Above and Left of each matrix). (B) Pie chart of the 6,398 ERG ChIP-seq peaks corresponding to the distribution of ERG binding relative to the nearest gene in RPWE1-ERG cells. Binding was broken down into the following four categories: within promoters (±2 kb from transcription start sites), intronic, 3′ from the end of the last exon (3′ End), and distal (>2 kb away from known genes). (C) Graph showing the amount of ERG binding (kb) at each of the ranked (N) differential interacting chromatin loci of 1-Mb regions plus a 1-Mb region flanking either side (3 Mb total window size) showing the top interaction loci are strongly enriched with ERG binding sites.

ChIP-seq, RNA-seq, and Hi-C data integration. (A) Network of genes (visualized using Cytoscape) (35) that are represented in independent cis-interactions between 1-Mb chromosome loci on chromosome 6 and that are significantly differentially expressed between RWPE-GFP (Left) and RWPE-ERG (Right) cells. Node color represents over- (red) and underexpressed (green) genes in RWPE-ERG cells relative to RWPE-GFP cells. Pink indicates that the genes in the node are both over- and underexpressed in RWPE-ERG cells. Node shapes are defined by ERG binding peak calls from ChIP-seq data [triangle, bound to promoter; diamond, bound to gene body; square, bound to distal region (between 2 kb and 50 kb); circle, bound to far distal region (> 50 kb)]. Edge (lines connecting the nodes) color represents interaction (from Hi-C) enriched in RWPE-ERG cells (orange) or RWPE-GFP cells (blue). (B and C) Validation of a subset of cis-interactions on chromosome 6 that involved ERG-regulated genes (MOXD1, HEY2, FYN, and SERPINB9). (B) Schematic map of chromosome 6 showing a subset of interactions and deregulated genes from A is shown with loci and anchor regions indicated. (C) (Left) The mean frequencies of interactions for the indicated loci pair based on PCR products obtained from two independent 3C libraries (above) and mRNA levels of the indicated genes (below) generated from either RWPE1-ERG (orange) or RPWE1-GFP (blue). SDs are shown as error bars. (Upper Right) Representative FISH images of G1-enriched nuclei based on analyses using probes made from BACs housing, to the left, MOXD1 (loci 1, red) and SERPINB9 (loci 6, green) or, to the right, FYN (loci 6, red) and HEY2 (loci 5, green), show close or distant proximity of the two loci. (Lower Right) Graphical representation of the percentage of nuclei having overlapping signals from RWPE1-ERG (orange bars) or RWPE1-GFP (blue bars) cells based on 251 and 304 (MOXD1-SERPINB9) nuclei or 150 and 150 nuclei (HEY2-FYN) evaluated, respectively (P value based on a two-tailed Fisher’s exact test is shown above).

Specific cis-interactions that depend on ERG expression. (A) Schematic representation of the experimental flow of the Hi-C experiments. Red symbols refer to ERG protein and white symbols refer to other DNA-interacting proteins. (B) Circos plot depicting chromosomes 1–22 and X annotated for ERG binding on the basis of ChIP-seq data (normalized reads shown in orange) and 1,266 differentially expressed genes annotated on the basis of RNA-seq data, indicating over- (red) and underexpressed genes in RWPE1-ERG relative to RWPE1-GFP cells. Lines indicate significantly different cis- and trans-interactions in which line color represents interactions enriched in RWPE-ERG cells (orange) or RWPE-GFP cells (blue).

(A) Pie chart of the 1,266 ERG-differentially expressed genes as a function of significantly interacting on the basis of the Hi-C data. These genes are divided into two groups: the interacting genes (housed in loci that are significantly interacting differently in the ERG compared with the control cell lines) and the noninteracting genes (housed in loci that are not significantly interacting differently in the ERG compared with the control cell lines). (B) Gene Ontology (GO) analysis results showing a subset of the significant categories (adjusted P value <0.05), using the interacting gene set. There were no significant categories based on the noninteracting gene set.

Validation of specific cis-interactions in DU145 prostate cancer cell lines overexpressing ERG alone or in the presence of shRNA targeting ERG mRNA. (A) ERG mRNA (Upper) and protein (Lower) levels measured by quantitative RT-PCR (17) and Western blot analysis in DU145 cells stably overexpressing ERG alone or in the presence of stable expression of shRNA targeting the ERG mRNA (two independent cell line clones are shown) or GFP as a control. shRNA expression led to a drastic reduction in transcript and protein levels. (B) Validation of a subset of cis-interactions on chromosome 6 that involved ERG-regulated genes (MOXD1, FYN, and SERPINB9) in the DU145 cell lines based on the primers described in the Fig. 3 legend (MOXD1 region is the anchor, and FYN regions are located 20.6 Mb and 129.96 Mb from the anchor region, respectively). (C) Validation of the overexpression of ERG-regulated genes (MOXD1, FYN, and SERPINB9) in the DU145 cell lines.