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RUNX1, an androgen- and EZH2-regulated gene, has differential roles in AR-dependent and -independent prostate cancer.

Takayama K, Suzuki T, Tsutsumi S, Fujimura T, Urano T, Takahashi S, Homma Y, Aburatani H, Inoue S - Oncotarget (2015)

Bottom Line: The RUNX1 promoter is bound by enhancer of zeste homolog 2 (EZH2) and is negatively regulated by histone H3 lysine 27 (K27) trimethylation.Repression of RUNX1 is important for the growth promotion ability of EZH2 in AR-independent cells.These results indicated the significance of RUNX1 for androgen-dependency and that loss of RUNX1 could be a key step for the progression of prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Anti-Aging Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
Androgen receptor (AR) signaling is essential for the development of prostate cancer. Here, we report that runt-related transcription factor (RUNX1) could be a key molecule for the androgen-dependence of prostate cancer. We found RUNX1 is a target of AR and regulated positively by androgen. Our RUNX1 ChIP-seq analysis indicated that RUNX1 is recruited to AR binding sites by interacting with AR. In androgen-dependent cancer, loss of RUNX1 impairs AR-dependent transcription and cell growth. The RUNX1 promoter is bound by enhancer of zeste homolog 2 (EZH2) and is negatively regulated by histone H3 lysine 27 (K27) trimethylation. Repression of RUNX1 is important for the growth promotion ability of EZH2 in AR-independent cells. In clinical prostate cancer samples, the RUNX1 expression level is negatively associated with EZH2 and that RUNX1 loss correlated with poor prognosis. These results indicated the significance of RUNX1 for androgen-dependency and that loss of RUNX1 could be a key step for the progression of prostate cancer.

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Genome-wide analysis of RUNX1 by ChIP-seq(A) Identification of RUNX1 binding sites by ChIP-seq. RUNX1 ChIP-seq analysis was performed in LNCaP cells. Cells were treated with vehicle or DHT for 24 h. Mapping of RUNX1 and AR binding sites in the vicinity of a representative androgen-regulated gene, TMPRSSS2 on chromosome 21. (B) RUNX1 binding sites (P < 10−5) were determined by MACS. (C) Overlapping of ARBSs with RUNX1 binding sites. Venn diagrams depict the overlap of significant ARBSs with RUNX1 binding sites. (D) Recruitment of RUNX1 to androgen receptor binding sites (ARBSs). LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using a RUNX1-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (E) Heat map of RUNX1 binding tag intensity around peaks of AR binding sites (F) Motif analysis of RUNX1 binding sequences demonstrated the enrichment of AR and collaborative factor motifs. We analyzed 200-bp DNA sequences around RUNX1 binding peaks by using HOMER. Top two motifs by this analysis are shown.
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Figure 2: Genome-wide analysis of RUNX1 by ChIP-seq(A) Identification of RUNX1 binding sites by ChIP-seq. RUNX1 ChIP-seq analysis was performed in LNCaP cells. Cells were treated with vehicle or DHT for 24 h. Mapping of RUNX1 and AR binding sites in the vicinity of a representative androgen-regulated gene, TMPRSSS2 on chromosome 21. (B) RUNX1 binding sites (P < 10−5) were determined by MACS. (C) Overlapping of ARBSs with RUNX1 binding sites. Venn diagrams depict the overlap of significant ARBSs with RUNX1 binding sites. (D) Recruitment of RUNX1 to androgen receptor binding sites (ARBSs). LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using a RUNX1-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (E) Heat map of RUNX1 binding tag intensity around peaks of AR binding sites (F) Motif analysis of RUNX1 binding sequences demonstrated the enrichment of AR and collaborative factor motifs. We analyzed 200-bp DNA sequences around RUNX1 binding peaks by using HOMER. Top two motifs by this analysis are shown.

Mentions: We investigated the genome-wide binding of RUNX1 in androgen-dependent transcriptional regulation by ChIP-seq. LNCaP cells were treated with vehicle or DHT for 24 h and then ChIP was performed using a RUNX1 specific antibody. Enriched DNA was sequenced using a next generation sequencer and sequenced tags were mapped to the human genome (Fig.2A). We obtained 1125 significant RUNX1 binding sites in vehicle-treated cells and 2040 in DHT-treated cells using MACS (ref. 21, P-value < 1.0E-5, Fig.2B). We have previously reported significant DHT-dependent ARBSs (defined by MACS P-value <1.0E−5) in a previous publication [20]. As we expected, overlap of ARBSs with RUNX1 binding sites was observed and we validated androgen-dependent RUNX1 recruitment to representative ARBSs (Fig.2C-D, Supplementary Fig.2A). In addition, analysis of global RUNX1 ChIP-seq signals has revealed the distribution of RUNX1 bindings around the peak centers of significant ARBSs (Fig. 2E). The enriched motifs of transcription factors in RUNX1 binding sites were analyzed using HOMER [22]. Interestingly, the canonical RUNX1 binding motifs were not enriched (0.5% of all binding sites) although FOXA1 and AR motifs were enriched (Fig.2F), suggesting indirect binding of RUNX1 in ARBSs by binding to AR.


RUNX1, an androgen- and EZH2-regulated gene, has differential roles in AR-dependent and -independent prostate cancer.

Takayama K, Suzuki T, Tsutsumi S, Fujimura T, Urano T, Takahashi S, Homma Y, Aburatani H, Inoue S - Oncotarget (2015)

Genome-wide analysis of RUNX1 by ChIP-seq(A) Identification of RUNX1 binding sites by ChIP-seq. RUNX1 ChIP-seq analysis was performed in LNCaP cells. Cells were treated with vehicle or DHT for 24 h. Mapping of RUNX1 and AR binding sites in the vicinity of a representative androgen-regulated gene, TMPRSSS2 on chromosome 21. (B) RUNX1 binding sites (P < 10−5) were determined by MACS. (C) Overlapping of ARBSs with RUNX1 binding sites. Venn diagrams depict the overlap of significant ARBSs with RUNX1 binding sites. (D) Recruitment of RUNX1 to androgen receptor binding sites (ARBSs). LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using a RUNX1-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (E) Heat map of RUNX1 binding tag intensity around peaks of AR binding sites (F) Motif analysis of RUNX1 binding sequences demonstrated the enrichment of AR and collaborative factor motifs. We analyzed 200-bp DNA sequences around RUNX1 binding peaks by using HOMER. Top two motifs by this analysis are shown.
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Figure 2: Genome-wide analysis of RUNX1 by ChIP-seq(A) Identification of RUNX1 binding sites by ChIP-seq. RUNX1 ChIP-seq analysis was performed in LNCaP cells. Cells were treated with vehicle or DHT for 24 h. Mapping of RUNX1 and AR binding sites in the vicinity of a representative androgen-regulated gene, TMPRSSS2 on chromosome 21. (B) RUNX1 binding sites (P < 10−5) were determined by MACS. (C) Overlapping of ARBSs with RUNX1 binding sites. Venn diagrams depict the overlap of significant ARBSs with RUNX1 binding sites. (D) Recruitment of RUNX1 to androgen receptor binding sites (ARBSs). LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using a RUNX1-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (E) Heat map of RUNX1 binding tag intensity around peaks of AR binding sites (F) Motif analysis of RUNX1 binding sequences demonstrated the enrichment of AR and collaborative factor motifs. We analyzed 200-bp DNA sequences around RUNX1 binding peaks by using HOMER. Top two motifs by this analysis are shown.
Mentions: We investigated the genome-wide binding of RUNX1 in androgen-dependent transcriptional regulation by ChIP-seq. LNCaP cells were treated with vehicle or DHT for 24 h and then ChIP was performed using a RUNX1 specific antibody. Enriched DNA was sequenced using a next generation sequencer and sequenced tags were mapped to the human genome (Fig.2A). We obtained 1125 significant RUNX1 binding sites in vehicle-treated cells and 2040 in DHT-treated cells using MACS (ref. 21, P-value < 1.0E-5, Fig.2B). We have previously reported significant DHT-dependent ARBSs (defined by MACS P-value <1.0E−5) in a previous publication [20]. As we expected, overlap of ARBSs with RUNX1 binding sites was observed and we validated androgen-dependent RUNX1 recruitment to representative ARBSs (Fig.2C-D, Supplementary Fig.2A). In addition, analysis of global RUNX1 ChIP-seq signals has revealed the distribution of RUNX1 bindings around the peak centers of significant ARBSs (Fig. 2E). The enriched motifs of transcription factors in RUNX1 binding sites were analyzed using HOMER [22]. Interestingly, the canonical RUNX1 binding motifs were not enriched (0.5% of all binding sites) although FOXA1 and AR motifs were enriched (Fig.2F), suggesting indirect binding of RUNX1 in ARBSs by binding to AR.

Bottom Line: The RUNX1 promoter is bound by enhancer of zeste homolog 2 (EZH2) and is negatively regulated by histone H3 lysine 27 (K27) trimethylation.Repression of RUNX1 is important for the growth promotion ability of EZH2 in AR-independent cells.These results indicated the significance of RUNX1 for androgen-dependency and that loss of RUNX1 could be a key step for the progression of prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Anti-Aging Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
Androgen receptor (AR) signaling is essential for the development of prostate cancer. Here, we report that runt-related transcription factor (RUNX1) could be a key molecule for the androgen-dependence of prostate cancer. We found RUNX1 is a target of AR and regulated positively by androgen. Our RUNX1 ChIP-seq analysis indicated that RUNX1 is recruited to AR binding sites by interacting with AR. In androgen-dependent cancer, loss of RUNX1 impairs AR-dependent transcription and cell growth. The RUNX1 promoter is bound by enhancer of zeste homolog 2 (EZH2) and is negatively regulated by histone H3 lysine 27 (K27) trimethylation. Repression of RUNX1 is important for the growth promotion ability of EZH2 in AR-independent cells. In clinical prostate cancer samples, the RUNX1 expression level is negatively associated with EZH2 and that RUNX1 loss correlated with poor prognosis. These results indicated the significance of RUNX1 for androgen-dependency and that loss of RUNX1 could be a key step for the progression of prostate cancer.

Show MeSH
Related in: MedlinePlus