Limits...
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.

Show MeSH

Related in: MedlinePlus

Enrichment of EZH2 and histone modifications on ID4 promoter in prostate cancer cell lines and tissue on Chromatin immuno-precipitated (ChIP) DNAA: Enrichment of Pol II, EZH2, H3K27me3 and H3Ac on ID4 promoter in prostate cancer cell lines LNCaP, DU145 and C81 cells. The data is expressed (mean+SEM, n=3 in triplicate) as % of input. The statistical significance between enrichment (indicated by letters “a”, “b”, “c” and “d” corresponding to Pol II, EZH2, H3K27me3 and H3Ac respectively) is based on comparison with LNCaP cells (*: P<0.001). B: Similar to “A” above but the enrichment was performed on DNA isolated from FFPE cancer/ normal prostate tissue by laser capture micro-dissection. The number of Benign and prostate cancer samples were 5 each.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4196193&req=5

Figure 3: Enrichment of EZH2 and histone modifications on ID4 promoter in prostate cancer cell lines and tissue on Chromatin immuno-precipitated (ChIP) DNAA: Enrichment of Pol II, EZH2, H3K27me3 and H3Ac on ID4 promoter in prostate cancer cell lines LNCaP, DU145 and C81 cells. The data is expressed (mean+SEM, n=3 in triplicate) as % of input. The statistical significance between enrichment (indicated by letters “a”, “b”, “c” and “d” corresponding to Pol II, EZH2, H3K27me3 and H3Ac respectively) is based on comparison with LNCaP cells (*: P<0.001). B: Similar to “A” above but the enrichment was performed on DNA isolated from FFPE cancer/ normal prostate tissue by laser capture micro-dissection. The number of Benign and prostate cancer samples were 5 each.

Mentions: Chromatin immuno-precipitation experiments demonstrated that ID4 promoter is enriched with EZH2 in C81 (% input 0.11+0.024, P<0.001) and DU145 (0.23+0.0.052, P<0.001) cells as compared to LNCaP cells (EZH2: 0.03+0.004). The corresponding H3K27me3 enrichment was also higher in DU145 and C81 cells (0.36+0.084 and 0.24+0.065 in C81 and DU145 respectively) as compared to LNCaP cells (0.11+0.021). Decreased RNA polymerase II (PolA) occupancy in C81 (0.037+0.0054) and DU145 (0.116+0.022) as compared to LNCaP (0.36+0.061) is also consistent with ID4 transcription (Fig. 3A) in these three cell lines. Actively transcribed genes also commonly exhibit increased H3 acetylation as opposed to decreased H3K27me3. As expected, decreased H3 acetylation was observed in C81 (0.079+0.036, P<0.001) and DU145 (0.13+0.021, P<0.001) cells as compared to LNCaP cells (0.26+0.033) (Fig. 3A).


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

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

Enrichment of EZH2 and histone modifications on ID4 promoter in prostate cancer cell lines and tissue on Chromatin immuno-precipitated (ChIP) DNAA: Enrichment of Pol II, EZH2, H3K27me3 and H3Ac on ID4 promoter in prostate cancer cell lines LNCaP, DU145 and C81 cells. The data is expressed (mean+SEM, n=3 in triplicate) as % of input. The statistical significance between enrichment (indicated by letters “a”, “b”, “c” and “d” corresponding to Pol II, EZH2, H3K27me3 and H3Ac respectively) is based on comparison with LNCaP cells (*: P<0.001). B: Similar to “A” above but the enrichment was performed on DNA isolated from FFPE cancer/ normal prostate tissue by laser capture micro-dissection. The number of Benign and prostate cancer samples were 5 each.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196193&req=5

Figure 3: Enrichment of EZH2 and histone modifications on ID4 promoter in prostate cancer cell lines and tissue on Chromatin immuno-precipitated (ChIP) DNAA: Enrichment of Pol II, EZH2, H3K27me3 and H3Ac on ID4 promoter in prostate cancer cell lines LNCaP, DU145 and C81 cells. The data is expressed (mean+SEM, n=3 in triplicate) as % of input. The statistical significance between enrichment (indicated by letters “a”, “b”, “c” and “d” corresponding to Pol II, EZH2, H3K27me3 and H3Ac respectively) is based on comparison with LNCaP cells (*: P<0.001). B: Similar to “A” above but the enrichment was performed on DNA isolated from FFPE cancer/ normal prostate tissue by laser capture micro-dissection. The number of Benign and prostate cancer samples were 5 each.
Mentions: Chromatin immuno-precipitation experiments demonstrated that ID4 promoter is enriched with EZH2 in C81 (% input 0.11+0.024, P<0.001) and DU145 (0.23+0.0.052, P<0.001) cells as compared to LNCaP cells (EZH2: 0.03+0.004). The corresponding H3K27me3 enrichment was also higher in DU145 and C81 cells (0.36+0.084 and 0.24+0.065 in C81 and DU145 respectively) as compared to LNCaP cells (0.11+0.021). Decreased RNA polymerase II (PolA) occupancy in C81 (0.037+0.0054) and DU145 (0.116+0.022) as compared to LNCaP (0.36+0.061) is also consistent with ID4 transcription (Fig. 3A) in these three cell lines. Actively transcribed genes also commonly exhibit increased H3 acetylation as opposed to decreased H3K27me3. As expected, decreased H3 acetylation was observed in C81 (0.079+0.036, P<0.001) and DU145 (0.13+0.021, P<0.001) cells as compared to LNCaP cells (0.26+0.033) (Fig. 3A).

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