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CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2.

Seo WY, Jeong BC, Yu EJ, Kim HJ, Kim SH, Lim JE, Kwon GY, Lee HM, Kim JH - Nucleic Acids Res. (2013)

Bottom Line: Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes.The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites.CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, 135-710, Korea, Department of Biomedical Sciences, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, 135-710, Korea, Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea and Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea.

ABSTRACT
Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer-promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis.

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CCAR1 is required for optimal association of AR with target enhancers and AR-mediated chromatin looping and chromatin remodeling. (A, B and C) LNCaP cells expressing shNS or shCCAR1 were treated with or without 10 nM DHT for 16 h. Protein levels were monitored by immunoblot using the indicated antibodies (A). ChIP assays using the indicated antibodies were performed as described in Figure 1C. qPCR analyses were performed using primers specific for the PSA (B) and TMPRSS2 (C) enhancers. The results are shown as percentage of input and are means ± SD (n = 3). (D) 3C assays were performed using crosslinked, BstYI-digested chromatin from LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. The ligated DNA was PCR amplified with primers as indicated. (E) FAIRE was conducted with LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. qPCR analyses were performed using primers specific for the PSA and TMPRSS2 enhancers.
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gkt644-F4: CCAR1 is required for optimal association of AR with target enhancers and AR-mediated chromatin looping and chromatin remodeling. (A, B and C) LNCaP cells expressing shNS or shCCAR1 were treated with or without 10 nM DHT for 16 h. Protein levels were monitored by immunoblot using the indicated antibodies (A). ChIP assays using the indicated antibodies were performed as described in Figure 1C. qPCR analyses were performed using primers specific for the PSA (B) and TMPRSS2 (C) enhancers. The results are shown as percentage of input and are means ± SD (n = 3). (D) 3C assays were performed using crosslinked, BstYI-digested chromatin from LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. The ligated DNA was PCR amplified with primers as indicated. (E) FAIRE was conducted with LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. qPCR analyses were performed using primers specific for the PSA and TMPRSS2 enhancers.

Mentions: Recently, we have reported that CCAR1 plays an important role in transcription complex assembly on target promoters by providing a physical link between activated transcription factors and Mediator complexes and thereby facilitates recruitment of Pol II to the promoter of target genes (8). To further test the role of CCAR1 as an AR co-activator, we assessed the effect of reduced CCAR1 levels on transcription complex assembly on the PSA enhancer. Reduction of CCAR1 levels by shRNA had no measurable effect on the cellular levels of AR, MED1 and Pol II (Figure 4A). However, CCAR1 depletion severely affected the DHT-dependent recruitment of AR to the PSA enhancer, and the recruitment of MED1 and Pol II was greatly reduced (Figure 4B). Similar results were observed on the TMPRSS2 and KLK2 enhancers (Figure 4C and Supplementary Figure S11A). Interestingly, CCAR1 depletion also abolished RNA Pol II recruitment to the PSA promoter but caused only a modest reduction in AR occupancy on the promoter (Supplementary Figure S12). The hormone-induced level of histone H3 acetylation was also reduced by CCAR1 depletion (Figure 4B and C and Supplementary Figure S11). These results are consistent with the data that CCAR1 is required for AR-mediated transcription (Figure 2) and strongly suggest that CCAR1 is required for efficient binding of AR to its regulatory regions and thereby facilitates the occupancy of Mediator and Pol II on the regulatory regions of AR target genes.Figure 4.


CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2.

Seo WY, Jeong BC, Yu EJ, Kim HJ, Kim SH, Lim JE, Kwon GY, Lee HM, Kim JH - Nucleic Acids Res. (2013)

CCAR1 is required for optimal association of AR with target enhancers and AR-mediated chromatin looping and chromatin remodeling. (A, B and C) LNCaP cells expressing shNS or shCCAR1 were treated with or without 10 nM DHT for 16 h. Protein levels were monitored by immunoblot using the indicated antibodies (A). ChIP assays using the indicated antibodies were performed as described in Figure 1C. qPCR analyses were performed using primers specific for the PSA (B) and TMPRSS2 (C) enhancers. The results are shown as percentage of input and are means ± SD (n = 3). (D) 3C assays were performed using crosslinked, BstYI-digested chromatin from LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. The ligated DNA was PCR amplified with primers as indicated. (E) FAIRE was conducted with LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. qPCR analyses were performed using primers specific for the PSA and TMPRSS2 enhancers.
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gkt644-F4: CCAR1 is required for optimal association of AR with target enhancers and AR-mediated chromatin looping and chromatin remodeling. (A, B and C) LNCaP cells expressing shNS or shCCAR1 were treated with or without 10 nM DHT for 16 h. Protein levels were monitored by immunoblot using the indicated antibodies (A). ChIP assays using the indicated antibodies were performed as described in Figure 1C. qPCR analyses were performed using primers specific for the PSA (B) and TMPRSS2 (C) enhancers. The results are shown as percentage of input and are means ± SD (n = 3). (D) 3C assays were performed using crosslinked, BstYI-digested chromatin from LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. The ligated DNA was PCR amplified with primers as indicated. (E) FAIRE was conducted with LNCaP cells expressing shNS or shCCAR1 treated with or without 10 nM DHT for 16 h. qPCR analyses were performed using primers specific for the PSA and TMPRSS2 enhancers.
Mentions: Recently, we have reported that CCAR1 plays an important role in transcription complex assembly on target promoters by providing a physical link between activated transcription factors and Mediator complexes and thereby facilitates recruitment of Pol II to the promoter of target genes (8). To further test the role of CCAR1 as an AR co-activator, we assessed the effect of reduced CCAR1 levels on transcription complex assembly on the PSA enhancer. Reduction of CCAR1 levels by shRNA had no measurable effect on the cellular levels of AR, MED1 and Pol II (Figure 4A). However, CCAR1 depletion severely affected the DHT-dependent recruitment of AR to the PSA enhancer, and the recruitment of MED1 and Pol II was greatly reduced (Figure 4B). Similar results were observed on the TMPRSS2 and KLK2 enhancers (Figure 4C and Supplementary Figure S11A). Interestingly, CCAR1 depletion also abolished RNA Pol II recruitment to the PSA promoter but caused only a modest reduction in AR occupancy on the promoter (Supplementary Figure S12). The hormone-induced level of histone H3 acetylation was also reduced by CCAR1 depletion (Figure 4B and C and Supplementary Figure S11). These results are consistent with the data that CCAR1 is required for AR-mediated transcription (Figure 2) and strongly suggest that CCAR1 is required for efficient binding of AR to its regulatory regions and thereby facilitates the occupancy of Mediator and Pol II on the regulatory regions of AR target genes.Figure 4.

Bottom Line: Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes.The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites.CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, 135-710, Korea, Department of Biomedical Sciences, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, 135-710, Korea, Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea and Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea.

ABSTRACT
Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer-promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis.

Show MeSH
Related in: MedlinePlus