Polycomb group genes are targets of aberrant DNA methylation in renal cell carcinoma

The combined effects of genetic and epigenetic aberrations are well recognized as causal in tumorigenesis. Here, we defined profiles of DNA methylation in primary renal cell carcinomas (RCC) and assessed the association of these profiles with the expression of genes required for the establishment and maintenance of epigenetic marks. A bead-based methylation array platform was used to measure methylation of 1,413 CpG loci in ∼800 cancer-associated genes and three methylation classes were derived by unsupervised clustering of tumors using recursively partitioned mixture modeling (RPMM). Quantitative RT-PCR was performed on all tumor samples to determine the expression of DNMT1, DNMT3B, VEZF1 and EZH2. Additionally, methylation at LINE-1 and AluYb8 repetitive elements was measured using bisulfite pyrosequencing. Associations between methylation class and tumor stage (p = 0.05), LINE-1 (p < 0.0001) and AluYb8 (p < 0.0001) methylation, as well as EZH2 expression (p < 0.0001) were noted following univariate analyses. A multinomial logistic regression model controlling for potential confounders revealed that AluYb8 (p < 0.003) methylation and EZH2 expression (p < 0.008) were significantly associated with methylation class membership. Because EZH2 is a member of the Polycomb repressive complex 2 (PRC2), we next analyzed the distribution of Polycomb group (PcG) targets among methylation classes derived by clustering the 1,413 array CpG loci using RPMM. PcG target genes were significantly enriched (p < 0.0001) in methylation classes with greater differential methylation between RCC and non-diseased kidney tissue. This work contributes to our understanding of how repressive marks on DNA and chromatin are dysregulated in carcinogenesis, knowledge that might aid the development of therapies or preventive strategies for human malignancies.Key words: EZH2, DNA methylation, renal cell carcinoma, polycomb, microarray     

Polycomb group genes are targets of aberrant DNA methylation in renal cell carcinoma

The combined effects of genetic and epigenetic aberrations are well recognized as causal in tumorigenesis. Here, we defined profiles of DNA methylation in primary renal cell carcinomas (RCC) and assessed the association of these profiles with the expression of genes required for the establishment and maintenance of epigenetic marks. A bead-based methylation array platform was used to measure methylation of 1,413 CpG loci in ~800 cancer-associated genes and three methylation classes were derived by unsupervised clustering of tumors using recursively partitioned mixture modeling (RPMM). Quantitative RT-PCR was performed on all tumor samples to determine the expression of DNMT1, DNMT3B, VEZF1 and EZH2. Additionally, methylation at LINE-1 and AluYb8 repetitive elements was measured using bisulfite pyrosequencing. Associations between methylation class and tumor stage (p = 0.05), LINE-1 (p &lt; 0.0001) and AluYb8 (p &lt; 0.0001) methylation, as well as EZH2 expression (p &lt; 0.0001) were noted following univariate analyses. A multinomial logistic regression model controlling for potential confounders revealed that AluYb8 (p &lt; 0.003) methylation and EZH2 expression (p &lt; 0.008) were significantly associated with methylation class membership. Because EZH2 is a member of the Polycomb repressive complex 2 (PRC2), we next analyzed the distribution of Polycomb group (PcG) targets among methylation classes derived by clustering the 1,413 array CpG loci using RPMM. PcG target genes were significantly enriched (p &lt; 0.0001) in methylation classes with greater differential methylation between RCC and non-diseased kidney tissue. This work contributes to our understanding of how repressive marks on DNA and chromatin are dysregulated in carcinogenesis, knowledge that might aid the development of therapies or preventive strategies for human malignancies.     

Physical Interaction between MYCN Oncogene and Polycomb Repressive Complex 2 (PRC2) in Neuroblastoma: FUNCTIONAL AND THERAPEUTIC IMPLICATIONS*

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.