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EZH2 dependent H3K27me3 is involved in epigenetic silencing of ID4 in prostate cancer.

Chinaranagari S, Sharma P, Chaudhary J - Oncotarget (2014)

Bottom Line: Here, we demonstrate that ID4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3).Enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines compared to ID4 expressing LNCaP cell line.Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA.

ABSTRACT
Inhibitor of DNA binding/differentiation protein 4 (ID4) is dominant negative helix loop helix transcriptional regulator is epigenetically silenced due to promoter hyper-methylation in many cancers including prostate. However, the underlying mechanism involved in epigenetic silencing of ID4 is not known. Here, we demonstrate that ID4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP data on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2 but decreased ID4 expression in prostate cancer strongly supports this model.

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Effect of EZH2 silencing on ID4 expressionA: Two different siRNAs (siRNA1 and siRNA2) were used to transiently knock down EZH2 in DU145 cells followed by western blot analysis of EZH2, ID4 and KLF2 (used as a positive control for EZH2 dependent down-regulated gene). Increase in ID4 expression with greater EZH2 knock down was observed with siRNA2 that was used for all subsequent studies. Representative western blot is shown. B: Real time quantitative RT-PCR analysis of corresponding gene expression following EZH2 knockdown by siRNA2. The data is expressed (mean+SEM, n=3 in triplicate) as fold change compared to non-specific siRNA. C: Immuno-cytochemical analysis of ID4 expression following siRNA2 mediated knockdown of EZH2 in DU145 cells (x200 magnification). ID4 expression is in red and the nuclei in Blue (DAPI). C.1 and C.4 are merged images of Blue (Nuclei, C.2 and C.5) and Red (ID4, C.3 and C.6). C.1, C.2 and C.3 are DU145 cells transfected with non-specific siRNA (DU145+NS). Panels C.4, C.5 and C.6 are DU145 cells transfected with EZH2 siRNA (DU145+siEZH2). Representative images are shown. D: Enrichment of EZH2, H3K27me3, H3Ac and DNMT1 on ID4 and KLF2 promoters following EZH2 knockdown in DU145 cells. The data is expressed (mean+SEM, n=3 in triplicate) fold change of % input as compared to DU145 cells transfected with non-specific EZH2 siRNA. E: Western blot analysis of DNMT1 expression in DU145 cells with non-silencing siRNA or with EZH2 si-RNA2 (siEZH2). Representative of 3 blots is shown. F: Methylation specific PCR (MSP) on ID4 promoter following knockdown of EZH2 in DU145 cells. A band in “M” lane represents methylation of ID4 promoter where as a band in “U” lane represents un-methylated promoter. Representative results are shown.
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Figure 4: Effect of EZH2 silencing on ID4 expressionA: Two different siRNAs (siRNA1 and siRNA2) were used to transiently knock down EZH2 in DU145 cells followed by western blot analysis of EZH2, ID4 and KLF2 (used as a positive control for EZH2 dependent down-regulated gene). Increase in ID4 expression with greater EZH2 knock down was observed with siRNA2 that was used for all subsequent studies. Representative western blot is shown. B: Real time quantitative RT-PCR analysis of corresponding gene expression following EZH2 knockdown by siRNA2. The data is expressed (mean+SEM, n=3 in triplicate) as fold change compared to non-specific siRNA. C: Immuno-cytochemical analysis of ID4 expression following siRNA2 mediated knockdown of EZH2 in DU145 cells (x200 magnification). ID4 expression is in red and the nuclei in Blue (DAPI). C.1 and C.4 are merged images of Blue (Nuclei, C.2 and C.5) and Red (ID4, C.3 and C.6). C.1, C.2 and C.3 are DU145 cells transfected with non-specific siRNA (DU145+NS). Panels C.4, C.5 and C.6 are DU145 cells transfected with EZH2 siRNA (DU145+siEZH2). Representative images are shown. D: Enrichment of EZH2, H3K27me3, H3Ac and DNMT1 on ID4 and KLF2 promoters following EZH2 knockdown in DU145 cells. The data is expressed (mean+SEM, n=3 in triplicate) fold change of % input as compared to DU145 cells transfected with non-specific EZH2 siRNA. E: Western blot analysis of DNMT1 expression in DU145 cells with non-silencing siRNA or with EZH2 si-RNA2 (siEZH2). Representative of 3 blots is shown. F: Methylation specific PCR (MSP) on ID4 promoter following knockdown of EZH2 in DU145 cells. A band in “M” lane represents methylation of ID4 promoter where as a band in “U” lane represents un-methylated promoter. Representative results are shown.

Mentions: To determine whether EZH2 in fact down-regulates ID4 expression directly, we performed RNA interference mediated knockdown of EZH2 in DU145 cells (DU145+siEZH2). The immuno blot analysis in DU145 cells showed knockdown of EZH2 with siRNA2 to a greater extent as compared to siRNA1 and non-specific siRNA (ns-siRNA) 72h after transfection (Fig. 4A). Based on these results, all subsequent studies were performed with EZH2 siRNA2. The results from quantitative real time PCR revealed that a significant increase in the expression of ID4 and KLF2 (Fig. 4B) in DU145+siEZH2 cells as compared to non-silencing controls (DU145+siNS). KLF2 was used as a positive control which is down-regulated by EZH2 [35]. The immuno-fluorescence analysis also revealed increased ID4 expression in DU145+siEZH2 as compared to non-silencing controls (Fig. 4C4). Knockdown of EZH2 also resulted in decreased EZH2 specific H3k27me3 repressive marks with a corresponding increase in the enrichment of transcriptionally active H3 acetylation marks and RNA polymerase II on ID4 promoter (Fig. 4D).


EZH2 dependent H3K27me3 is involved in epigenetic silencing of ID4 in prostate cancer.

Chinaranagari S, Sharma P, Chaudhary J - Oncotarget (2014)

Effect of EZH2 silencing on ID4 expressionA: Two different siRNAs (siRNA1 and siRNA2) were used to transiently knock down EZH2 in DU145 cells followed by western blot analysis of EZH2, ID4 and KLF2 (used as a positive control for EZH2 dependent down-regulated gene). Increase in ID4 expression with greater EZH2 knock down was observed with siRNA2 that was used for all subsequent studies. Representative western blot is shown. B: Real time quantitative RT-PCR analysis of corresponding gene expression following EZH2 knockdown by siRNA2. The data is expressed (mean+SEM, n=3 in triplicate) as fold change compared to non-specific siRNA. C: Immuno-cytochemical analysis of ID4 expression following siRNA2 mediated knockdown of EZH2 in DU145 cells (x200 magnification). ID4 expression is in red and the nuclei in Blue (DAPI). C.1 and C.4 are merged images of Blue (Nuclei, C.2 and C.5) and Red (ID4, C.3 and C.6). C.1, C.2 and C.3 are DU145 cells transfected with non-specific siRNA (DU145+NS). Panels C.4, C.5 and C.6 are DU145 cells transfected with EZH2 siRNA (DU145+siEZH2). Representative images are shown. D: Enrichment of EZH2, H3K27me3, H3Ac and DNMT1 on ID4 and KLF2 promoters following EZH2 knockdown in DU145 cells. The data is expressed (mean+SEM, n=3 in triplicate) fold change of % input as compared to DU145 cells transfected with non-specific EZH2 siRNA. E: Western blot analysis of DNMT1 expression in DU145 cells with non-silencing siRNA or with EZH2 si-RNA2 (siEZH2). Representative of 3 blots is shown. F: Methylation specific PCR (MSP) on ID4 promoter following knockdown of EZH2 in DU145 cells. A band in “M” lane represents methylation of ID4 promoter where as a band in “U” lane represents un-methylated promoter. Representative results are shown.
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Figure 4: Effect of EZH2 silencing on ID4 expressionA: Two different siRNAs (siRNA1 and siRNA2) were used to transiently knock down EZH2 in DU145 cells followed by western blot analysis of EZH2, ID4 and KLF2 (used as a positive control for EZH2 dependent down-regulated gene). Increase in ID4 expression with greater EZH2 knock down was observed with siRNA2 that was used for all subsequent studies. Representative western blot is shown. B: Real time quantitative RT-PCR analysis of corresponding gene expression following EZH2 knockdown by siRNA2. The data is expressed (mean+SEM, n=3 in triplicate) as fold change compared to non-specific siRNA. C: Immuno-cytochemical analysis of ID4 expression following siRNA2 mediated knockdown of EZH2 in DU145 cells (x200 magnification). ID4 expression is in red and the nuclei in Blue (DAPI). C.1 and C.4 are merged images of Blue (Nuclei, C.2 and C.5) and Red (ID4, C.3 and C.6). C.1, C.2 and C.3 are DU145 cells transfected with non-specific siRNA (DU145+NS). Panels C.4, C.5 and C.6 are DU145 cells transfected with EZH2 siRNA (DU145+siEZH2). Representative images are shown. D: Enrichment of EZH2, H3K27me3, H3Ac and DNMT1 on ID4 and KLF2 promoters following EZH2 knockdown in DU145 cells. The data is expressed (mean+SEM, n=3 in triplicate) fold change of % input as compared to DU145 cells transfected with non-specific EZH2 siRNA. E: Western blot analysis of DNMT1 expression in DU145 cells with non-silencing siRNA or with EZH2 si-RNA2 (siEZH2). Representative of 3 blots is shown. F: Methylation specific PCR (MSP) on ID4 promoter following knockdown of EZH2 in DU145 cells. A band in “M” lane represents methylation of ID4 promoter where as a band in “U” lane represents un-methylated promoter. Representative results are shown.
Mentions: To determine whether EZH2 in fact down-regulates ID4 expression directly, we performed RNA interference mediated knockdown of EZH2 in DU145 cells (DU145+siEZH2). The immuno blot analysis in DU145 cells showed knockdown of EZH2 with siRNA2 to a greater extent as compared to siRNA1 and non-specific siRNA (ns-siRNA) 72h after transfection (Fig. 4A). Based on these results, all subsequent studies were performed with EZH2 siRNA2. The results from quantitative real time PCR revealed that a significant increase in the expression of ID4 and KLF2 (Fig. 4B) in DU145+siEZH2 cells as compared to non-silencing controls (DU145+siNS). KLF2 was used as a positive control which is down-regulated by EZH2 [35]. The immuno-fluorescence analysis also revealed increased ID4 expression in DU145+siEZH2 as compared to non-silencing controls (Fig. 4C4). Knockdown of EZH2 also resulted in decreased EZH2 specific H3k27me3 repressive marks with a corresponding increase in the enrichment of transcriptionally active H3 acetylation marks and RNA polymerase II on ID4 promoter (Fig. 4D).

Bottom Line: Here, we demonstrate that ID4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3).Enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines compared to ID4 expressing LNCaP cell line.Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA.

ABSTRACT
Inhibitor of DNA binding/differentiation protein 4 (ID4) is dominant negative helix loop helix transcriptional regulator is epigenetically silenced due to promoter hyper-methylation in many cancers including prostate. However, the underlying mechanism involved in epigenetic silencing of ID4 is not known. Here, we demonstrate that ID4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP data on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2 but decreased ID4 expression in prostate cancer strongly supports this model.

Show MeSH
Related in: MedlinePlus