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     

Association of BMI1 with polycomb bodies is dynamic and requires PRC2/EZH2 and the maintenance DNA methyltransferase DNMT1

Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized. Polycomb repressive complex 2 (PRC2) is thought to be involved in the initiation of gene silencing, whereas Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression. Here, we investigate the kinetic properties of the binding of one of the PRC1 core components, BMI1, with PcG bodies. PcG bodies are unique nuclear structures located on regions of pericentric heterochromatin, found to be the site of accumulation of PcG complexes in different cell lines. We report the presence of at least two kinetically different pools of BMI1, a highly dynamic and a less dynamic fraction, which may reflect BMI1 pools with different binding capacities to these stable heterochromatin domains. Interestingly, PRC2 members EED and EZH2 appear to be essential for BMI1 recruitment to the PcG bodies. Furthermore, we demonstrate that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity. Together, these results provide new insights in the mechanism for regulation of chromatin silencing by PcG proteins and suggest a highly regulated recruitment of PRC1 to chromatin.     

Targeting of EZH2 to a defined genomic site is sufficient for recruitment of Dnmt3a but not de novo DNA methylation

Polycomb-mediated gene silencing and DNA methylation underlie many epigenetic processes important in normal development as well as in cancer. An interaction between EZH2 of the Polycomb repressive complex 2 (PRC2), which trimethylates lysine 27 on Histone 3 (H3K27me3), and all three DNA methyltransferases (DNMTs) has been demonstrated, implicating a role for PRC2 in directing DNA methylation. Interestingly, however, the majority of H3K27me3 marked genes lack DNA methylation in ES cells, indicating that EZH2 recruitment may not be sufficient to promote DNA methylation. Here, we employed a Gal4DBD/gal4UAS-based system to directly test if EZH2 binding at a defined genomic site is sufficient to promote de novo DNA methylation in a murine erythroleukaemia cell line. Targeting of a Gal4DBD-EZH2 fusion to an intergenic transgene bearing a gal4 binding-site array promoted localized recruitment of SUZ12 and BMI1, subunits of PRC2 and PRC1, respectively, and deposition of H3K27me3. Further analysis of the H3K27me3-marked site revealed the persistence of H3K4me2, a mark inversely correlated with DNA methylation. Strikingly, while DNMT3a was also recruited in an EZH2-dependent manner, de novo DNA methylation of the transgene was not observed. Thus, while targeting of EZH2 to a specific genomic site is sufficient for recruitment of DNMT3a, additional events are required for de novo DNA methylation.     

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.     

Phosphoryl-EZH-ion

Polycomb group (PcG) proteins regulate gene expression in embryonic and adult stem cells, but the mechanisms responsible for PcG gene targeting and regulation remain largely unknown. Recent evidence shows that EZH2, the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), is a nuclear phosphoprotein linking cell-cycle-intrinsic or extracellular signals to specific epigenetic signatures.     

Breast Cancer DNA Methylation Profiles Are Associated with Tumor Size and Alcohol and Folate Intake

Although tumor size and lymph node involvement are the current cornerstones of breast cancer prognosis, they have not been extensively explored in relation to tumor methylation attributes in conjunction with other tumor and patient dietary and hormonal characteristics. Using primary breast tumors from 162 (AJCC stage I–IV) women from the Kaiser Division of Research Pathways Study and the Illumina GoldenGate methylation bead-array platform, we measured 1,413 autosomal CpG loci associated with 773 cancer-related genes and validated select CpG loci with Sequenom EpiTYPER. Tumor grade, size, estrogen and progesterone receptor status, and triple negative status were significantly (Q-values <0.05) associated with altered methylation of 209, 74, 183, 69, and 130 loci, respectively. Unsupervised clustering, using a recursively partitioned mixture model (RPMM), of all autosomal CpG loci revealed eight distinct methylation classes. Methylation class membership was significantly associated with patient race (P<0.02) and tumor size (P<0.001) in univariate tests. Using multinomial logistic regression to adjust for potential confounders, patient age and tumor size, as well as known disease risk factors of alcohol intake and total dietary folate, were all significantly (P<0.0001) associated with methylation class membership. Breast cancer prognostic characteristics and risk-related exposures appear to be associated with gene-specific tumor methylation, as well as overall methylation patterns.     

Arabidopsis MSI1 connects LHP1 to PRC2 complexes

Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1–MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication.     

Biological evaluation of tanshindiols as EZH2 histone methyltransferase inhibitors

EZH2 is the core subunit of Polycomb repressive complex 2 catalyzing the methylation of histone H3 lysine-27 and closely involved in tumorigenesis. To discover small molecule inhibitors for EZH2 methyltransferase activity, we performed an inhibitor screen with catalytically active EZH2 protein complex and identified tanshindiols as EZH2 inhibitors. Tanshindiol B and C potently inhibited the methyltransferase activity in in vitro enzymatic assay with IC₅₀ values of 0.52 μM and 0.55 μM, respectively. Tanshindiol C exhibited growth inhibition of several cancer cells including Pfeiffer cell line, a diffuse large B cell lymphoma harboring EZH2 A677G activating mutation. Tanshindiol treatment in Pfeiffer cells significantly decreased the tri-methylated form of histone H3 lysine-27, a substrate of EZH2, as revealed by Western blot analysis and histone methylation ELISA. Based on enzyme kinetics and docking studies, we propose that tanshindiol-mediated inhibition of EZH2 activity is competitive for the substrate S-adenosylmethionine. Taken together, our findings strongly suggest that tanshindiols possess a unique anti-cancer activity whose mechanism involves the inhibition of EZH2 activity and would provide chemically valuable information for designing a new class of potent EZH2 inhibitors.     

SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus

Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16(INK4a) induction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15(INK4b) and p16(INK4a), but not of p14(ARF). Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control.     

DNA methyltransferase controls stem cell aging by regulating BMI1 and EZH2 through microRNAs

Epigenetic regulation of gene expression is well known mechanism that regulates cellular senescence of cancer cells. Here we show that inhibition of DNA methyltransferases (DNMTs) with 5-azacytidine (5-AzaC) or with specific small interfering RNA (siRNA) against DNMT1 and 3b induced the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) and increased p16(INK4A) and p21(CIP1/WAF1) expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG islands in the p16(INK4A) and p21(CIP1/WAF1) promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, was decreased on the p16(INK4A) and p21(CIP1/WAF1) promoter regions. We found that DNMT inhibition decreased expression levels of Polycomb-group (PcG) proteins and increased expression of microRNAs (miRNAs), which target PcG proteins. Decreased CpG island methylation and increased levels of active histone marks at genomic regions encoding miRNAs were observed after 5-AzaC treatment. Taken together, DNMTs have a critical role in regulating the cellular senescence of hUCB-MSCs through controlling not only the DNA methylation status but also active/inactive histone marks at genomic regions of PcG-targeting miRNAs and p16(INK4A) and p21(CIP1/WAF1) promoter regions.