CHD1 Loss Alters AR Binding at Lineage-Specific Enhancers and Modulates Distinct Transcriptional Programs to Drive Prostate Tumorigenesis

Abstract

Deletion of the gene encoding the chromatin remodeler CHD1 is among the most common alterations in prostate cancer (PCa); however, the tumor-suppressive functions of CHD1 and reasons for its tissue-specific loss remain undefined. We demonstrated that CHD1 occupied prostate-specific enhancers enriched for the androgen receptor (AR) and lineage-specific cofactors. Upon CHD1 loss, the AR cistrome was redistributed in patterns consistent with the oncogenic AR cistrome in PCa samples and drove tumor formation in the murine prostate. Notably, this cistrome shift was associated with a unique AR transcriptional signature enriched for pro-oncogenic pathways unique to this tumor subclass. Collectively, these data credential CHD1 as a tumor suppressor in the prostate that constrains AR binding/function to limit tumor progression.

Keywords: AR; CHD1; HOXB13; androgen receptor; chromatin remodeling; cistrome reprogramming; epigenetics; interactome; prostate cancer; prostate cancer subclass.

Figure 1.

Genomic loss of Chdl drives progression of prostate carcinoma in vivo. (A) CHD1 homozygous deletions in the TCGA database (The Cancer Genome Atlas Research, 2015) (Histogram) and Weill Cornell Institute for Precision Medicine database (pie charts) for all available samples ((Pauli et al., 2017) and in preparation). (B) Schematic of inducible Chdl knockout in the murine prostate. (C) H&E staining of prostates from Chd1^+/+ and Chd1^f/f mice in a Pb-Cre background at 1 year (50 μm scalebar). (D) Schematic of castration-testosterone re-supplementation in Chd1^+/+ (n = 4) and Chd1^f/f (n = 6) mice (top) and quantification of Ki67 staining after testosterone (T-pellet) re-supplementation (bottom) (50 μm scalebar). Data represent +/− SEM. (E) H&E staining of murine prostates at 1 year (50 μm scalebar). (F) Quantification of pathological features per genotype at 1 year. (G) Prostatic mass from 1 year old mice (sum of all lobes). Boxes represent the mean and interquartile range, with min and max values indicated. See also Figure S1.

Figure 2.

CHD1 co-localizes to the promoters of actively transcribed genes in PCa models. (A) Overlap of CHD1, H3K4me3, and H3K27me3 ChIP Seq peaks from LNCaP cells under androgen proficient conditions. (B) Representative binding of each factor on chromatin. (C) Heatmap of binding patterns for each factor from the Transcriptional Start Site (TSS) to the Transcriptional End Site (TES) of ~21000 RefSeq genes (gene body scaled to 3 kb, 500 bp before/after TSS/TES). (D) Average signal of H3K4me3 and CHD1 centered at the TSS of co-bound genes (normalized by RPKM). (E) Signal of CHD1 at all H3K4me3 or H3K27me3 peaks. (F) Ranked normalized RNA Seq transcripts obtained from androgen proficient conditions (GSE43785 (Chen et al., 2015)) ordered from most abundant (top) to least abundant (bottom), and the signal of CHD1, H3K4me3, and H3K27me3 are plotted for each gene centered around the TSS. 2-tailed spearman correlations are reported for each comparison; p < 0.0001 for all correlations. See also Figure S2.

Figure 3.

The CHD1 interactome is enriched for AR cofactors. (A) RIME was performed for H3K4me3 and CHD1 in androgen proficient conditions. Signal to noise ratios of significantly enriched peptides from H3K4me3 were subtracted from those of CHD1 and plotted by biological replicate. Orange: peptides unique to H3K4me3; Blue: unique to CHD1; Purple: common. Green: overlap with known AR interactors (Gottlieb et al., 2012; Mooslehner et al., 2012). (B) STRING analysis of AR (Stelloo et al., 2018a) and CHD1 interactomes in LNCaP cells. (C) Proximity Ligation Assay (PLA) for AR and CHD1 in LNCaP cells (left) and quantification of PLA signal from a single plane, plotted as the number of detected foci per cell (right). One-way Anova, Tukey’s multiple comparisons test: ** p < 0.01, **** p < 0.00001. Data represent +/− SEM. Scalebar 20 μm. See also Table S1–S2.

Figure 4.

CHD1 colocalizes to enhancers enriched for AR and its cofactors. (A) Overlap of CHD1 and H3K4me3 ChIP Seq peaks in LNCaP cells under androgen proficient conditions (top) and annotation of the common and unique peaks for H3K4me3 and CHD1 plotted as a percentage of all peaks (bottom). (B) Motif analysis of CHD1 peaks binned into promoter/TSS or intronic/intergenic categories (200 bp window). (C) Overlap of CHD1, AR, and H3K4me3 ChIP Seq peaks (top) and snapshot of AR, CHD1, and H3K4me3 signal at the KLK3 locus (bottom). (D) Overlap between CHD1 and AR, HOXB13 (Pomerantz et al., 2015), ETV1 (Chen et al., 2013), or FOXA1 (Zhao et al., 2016) ChIP Seq peaks in LNCaP cells under androgen-proficient conditions (top) and normalized average signal profile of CHD1 and each transcription factor at the peak center (bottom). See also Figure S3.

Figure 5.

AR binding is redistributed in the absence of CHD1. (A) Immunoblot (top) and immunofluorescence (bottom) of LNCaP CRISPR-Cas9 cell lines with Ctrl or CHD1 specific sgRNA. Scale bar 20 μm. (B) Growth curve of sgCtrl (left) and sgCHD1 cells (right) in response to increasing doses of dihydrotestosterone (DHT). Data represents +/− SEM. (C) ChIP Seq of differential AR binding between sgCtrl and sgCHD1. (D) RPKM normalized AR signal (sgCtrl signal subtracted from sgCHD1 signal) at sites enriched or depleted with CHD1 loss (left) and snapshots of sgCHD1 enriched and depleted AR peaks (right). (E) AR ChIP Seq from (GSE70079 (Pomerantz et al., 2015)) patient tumors centered at sgCHD1 enriched and depleted sites. (F) H3K27ac ChIP Seq from annotated primary PCa tumors (ERR3 59744 (Kron et al., 2017)) binned into CHD1 null and CHD1 WT categories. Average signal is plotted at sgCHD1 enriched and depleted sites. See also Figures S4 and S5.

Figure 6.

AR is enriched at HOXB13 sites in CHD1 null tumors. (A) Heatmap of differential motif enrichment between sgCHD1 enriched and depleted sites (left) and histogram of the average incidence of the HOXB13 (top) and AR-Halfsite (bottom) motifs from the peak center (right). (B) ChIP-seq for HOXB13 derived from LNCaP and primary human PCa (GSE70079 (Pomerantz et al., 2015)), was plotted at sgCHD1 enriched and depleted AR sites. (C) Average ChIP-seq signal for AR in control (left) and HOXB13 expressing (right) LHSAR cells at sgCHD1 enriched and depleted AR sites (GSE70079 (Pomerantz et al., 2015)). (D) AR ChIP Seq peaks from primary human prostate tumors (Stelloo et al., 2018b) binned into CHD1 deficient and CHD1 WT categories, merged, and assessed for overlap. (E) De novo motif analysis for CHD1 deficient tumor peaks (compared to CHD1 WT tumors) and enriched (blue) and de-enriched (red) motifs plotted according to rank and p value. Values next to motifs represent the best match motif score (out of 1). Gray indicates motifs with less significant p values. (F) ATAC-seq in the sgCHD1 isogenic model +/− 4 hr of DHT. 20k peaks/condition were randomly assessed for ARE and HOXB13 motif enrichment 100x. Plotted are the number of ARE (top) and HOXB13 (bottom) motifs per 20k peaks (left) and histogram of mean ARE and HOXB13 motif enrichments under DHT conditions (right). Data represent +/− SEM. (G) Immunoblot of LNCaP cells expressing HOXB13 or vector (left) and cell growth in androgen-depleted media in response to DHT (bottom). Data represent +/− SEM. See also Figure S6 and Table S3.

Figure 7.

Loss of CHD1 drives a subtype specific AR transcriptome associated with activation of oncogenic pathways. (A) 3D representation of GSEA Hallmark enrichment for 3 independent datasets deficient in CHD1. Normalized Enrichment Scores (NES) for all hallmarks for each CHD1 null model (vs. WT counterpart) are plotted on the X (LNCaP), Y (murine organoid), and Z (CHD1 null human tumors) axes. Hallmarks with consistent de-enrichment (blue) or enrichment (red) are highlighted (boxes). Gray dots represent inconsistent enrichment patterns. Individual NES’s for each highlighted hallmark and dataset are shown. (B) TCGA RNA-seq data was annotated for the status of ERG, ETS, and CHD1 and AR score plotted for each tumor. The box shows interquartile range with median (solid line) and min and max (whiskers) indicated. (C) Z-scores of all genes in the AR score were averaged per subclass and plotted by rank. Highlighted: The 3 top (red) and bottom (blue) genes contributing to the CHD1 null AR score. (D) RNA-seq from sgCtrl and sgCHDl cells stimulated with androgen for 0, 3, or 8 hr. Normalized z-scores of AR score genes for each model. Diminished and elevated genes have a p value of < 0.05 and fold change cutoff of 1.3x. (E) Ranked normalized z-scores of AR score genes from the sgCHD1 model. Average z-scores from TCGA tumor subclasses and sgCtrl were used to cluster datasets based on the sgCHD1 ranked list. (F) AR signature of sgCHD1 cells. Adjusted p value < 0.05. (G) NES’s for each subclass were generated for the upregulated (orange up arrows) and downregulated (purple down arrows) genes of the sgCHDl AR signature. The downregulated NES was subtracted from the upregulated NES for each genotype. (H) Metascape analysis of the sgCHDl AR signature. Red: Relevant to PCa. Blue: Consistent with normal prostate function. Purple: Common. See also Figure S7 and Table S4.