Methylation Silencing Research Articles

Novel epigenetic regulation of skeletal muscle myosin heavy chain genes. Focus on “Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading”

skeletal muscle is a highly plastic tissue, capable of responding and adapting to a variety of physiological stimuli, e.g., muscle loading and unloading. Multiple functional phenotypes of skeletal muscle exist throughout the body at the same time. For example, muscles required for antigravity support of the skeleton or sustained locomotion are generally slow-contracting, low force-generating, and relatively resistant to fatigue, while other muscles required for quick, powerful movements are generally fast-contracting, high force-generating, and easily fatigued. In fact, skeletal muscle phenotypes have been characterized using combinations of muscle properties, including metabolic, fatigability, color, innervation, or predominant contractile protein expression. Myosin heavy chain (MHC) is the most abundant protein in skeletal muscle, comprising ∼25% of the total protein content, and is a major component of the complex responsible for generating contractile force in skeletal muscle. At least nine MHC isoforms, each transcribed from their own gene, exist in mammalian striated muscle; but only slow type I MHC and various isoforms of fast type II (IIa, IIx/d, and IIb MHC isoforms) are present in adult limb skeletal muscle that is not undergoing regeneration (16). It is well established that skeletal muscle phenotype, particularly MHC gene expression, can be altered by different states of muscular activity or inactivity, as well as various hormonal and metabolic factors (1).

Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

Methylation of CLDN6, FBN2, RBP1, RBP4, TFPI2, and TMEFF2 in esophageal squamous cell carcinoma

In the development and progression of cancer, tumor suppressor genes may be silenced by mechanisms such as methylation. Thus the discovery of new genes silenced by methylation may uncover new tumor suppressor genes, and improve our understanding of cancer biology. In this study we investigated the methylation of 19 genes in esophageal squamous cell carcinoma. Methylation was measured in 10 of these genes in esophageal squamous cell carcinoma cell lines: CDH13, CLDN6, C16orf62, FBN2, FNBP1, ID4, RBP1, RBP4, TFPI2 and TMEFF2. To determine if there was a correlation between DNA methylation and gene silencing, each cell line was cultured with or without the demethylating drug 5-aza-2'-deoxycytidine (aza-dC). For 6 genes (CLDN6, FBN2, RBP1, RBP4, TFPI2 and TMEFF2) there was an association between reduction of methylation and increase in mRNA expression in the demethylated cell lines. The frequency of the methylation of these 6 genes in esophageal squamous cell carcinoma resection specimens was also investigated. All 6 genes showed more frequent methylation in the tumor than the matched proximal resection margin of uninvolved esophagus. There was a significant difference in the frequency of methylation and in the extent of the methylation between the cancer and the margin tissues for CLDN6, FBN2, TFPI2 and TMEFF2 (P=0.0007, P=0.0048 P=0.0002 and P<0.0001, respectively). This is the first report of gene silencing by methylation of CLDN6, FBN2, RBP4, TFPI2 and TMEFF2 in esophageal squamous cell carcinoma.

Combined inhibition of DNA methylation and histone acetylation enhances gene re-expression and drug sensitivity in vivo

Histone deacetylation and DNA methylation have a central role in the control of gene expression in tumours, including transcriptional repression of tumour suppressor genes and genes involved in sensitivity to chemotherapy. Treatment of cisplatin-resistant cell lines with an inhibitor of DNA methyltransferases, 2-deoxy-5′azacytidine (decitabine), results in partial reversal of DNA methylation, re-expression of epigenetically silenced genes including hMLH1 and sensitisation to cisplatin both in vitro and in vivo. We have investigated whether the combination of decitabine and a clinically relevant inhibitor of histone deacetylase activity (belinostat, PXD101) can further increase the re-expression of genes epigenetically silenced by DNA methylation and enhance chemo-sensitisation in vivo at well-tolerated doses. The cisplatin-resistant human ovarian cell line A2780/cp70 has the hMLH1 gene methylated and is resistant to cisplatin both in vitro and when grown as a xenograft in mice. Treatment of A2780/cp70 with decitabine and belinostat results in a marked increase in expression of epigenetically silenced MLH1 and MAGE-A1 both in vitro and in vivo when compared with decitabine alone. The combination greatly enhanced the effects of decitabine alone on the cisplatin sensitivity of xenografts. As the dose of decitabine that can be given to patients and hence the maximum pharmacodynamic effect as a demethylating agent is limited by toxicity and eventual re-methylation of genes, we suggest that the combination of decitabine and belinostat could have a role in the efficacy of chemotherapy in tumours that have acquired drug resistance due to DNA methylation and gene silencing.

CpG Island Tumor Suppressor Promoter Methylation in Non-BRCA-Associated Early Mammary Carcinogenesis

Only 5% of all breast cancers are the result of BRCA1/2 mutations. Methylation silencing of tumor suppressor genes is well described in sporadic breast cancer; however, its role in familial breast cancer is not known. CpG island promoter methylation was tested in the initial random periareolar fine-needle aspiration sample from 109 asymptomatic women at high risk for breast cancer. Promoter methylation targets included RARB (M3 and M4), ESR1, INK4a/ARF, BRCA1, PRA, PRB, RASSF1A, HIN-1, and CRBP1. Although the overall frequency of CpG island promoter methylation events increased with age (P<0.0001), no specific methylation event was associated with age. In contrast, CpG island methylation of RARB M4 (P=0.051), INK4a/ARF (P=0.042), HIN-1 (P=0.044), and PRA (P=0.032), as well as the overall frequency of methylation events (P=0.004), was associated with abnormal Masood cytology. The association between promoter methylation and familial breast cancer was tested in 40 unaffected premenopausal women in our cohort who underwent BRCA1/2 mutation testing. Women with BRCA1/2 mutations had a low frequency of CpG island promoter methylation (15 of 15 women had <or=4 methylation events), whereas women without a mutation showed a high frequency of promoter methylation events (24 of 25 women had 5-8 methylation events; P<0.0001). Of women with a BRCA1/2 mutation, none showed methylation of HIN-1 and only 1 of 15 women showed CpG island methylation of RARB M4, INK4a/ARF, or PRB promoters. This is the first evidence of CpG island methylation of tumor suppressor gene promoters in non-BRCA1/2 familial breast cancer.

Distinct BRAF (V600E) and KRAS Mutations in High Microsatellite Instability Sporadic Colorectal Cancer in African Americans

Colorectal cancer develops through genetic, epigenetic, and environmental events that result in uncontrolled cell proliferation. Colorectal cancer incidence and mortality is higher in African Americans (AA) than in the general population. Here, we carried out a molecular analysis of sporadic colorectal cancer tumors from AAs to investigate possible explanations for the observed disparities. A total of 222 AA colorectal cancer tumors were analyzed for microsatellite instability (MSI) for protein expression of two DNA mismatch repair genes, MLH1 and MSH2, by immunohistochemistry; for the methylation silencing of MLH1, p16, APC, and APC2 promoters by methylation-specific PCR; and for point mutations in two oncogenes, KRAS and BRAF, by sequencing. In our sample, 19.8% of the AAs colorectal cancer tumors were MSI high (MSI-H) and did not associate with any of the clinicopathologic features, except tumor differentiation. Higher levels of inactive DNA mismatch repair proteins MLH1 (41%) and MSH2 (33%) were found by immunohistochemistry. Methylation-specific PCR analysis revealed a high level of methylation for MLH1 (66%), APC (53%), and APC2 (90%), but not for p16 (26%). BRAF mutations were only within the MSI-H tumors, whereas most (64%) of KRAS mutations were found within the non-MSI-H group. MLH1, MSH2, and BRAF alterations are significantly associated with MSI-H phenotype, unlike APC, APC2 and KRAS alterations. The prominent role of DNA mismatch repair gene suppression in MSI-H and a distinctive role of BRAF and KRAS mutations with respect to MSI status are supported by this study.

DLEC1 is a functional 3p22.3 tumour suppressor silenced by promoter CpG methylation in colon and gastric cancers

Promoter CpG methylation of tumour suppressor genes (TSGs) is an epigenetic biomarker for TSG identification and molecular diagnosis. We screened genome wide for novel methylated genes through methylation subtraction of a genetic demethylation model of colon cancer (double knockout of DNMT1 and DNMT3B in HCT116) and identified DLEC1 (Deleted in lung and oesophageal cancer 1), a major 3p22.3 TSG, as one of the methylated targets. We further found that DLEC1 was downregulated or silenced in most colorectal and gastric cell lines due to promoter methylation, whereas broadly expressed in normal tissues including colon and stomach, and unmethylated in expressing cell lines and immortalised normal colon epithelial cells. DLEC1 expression was reactivated through pharmacologic or genetic demethylation, indicating a DNMT1/DNMT3B-mediated methylation silencing. Aberrant methylation was further detected in primary colorectal (10 out of 34, 29%) and gastric tumours (30 out of 89, 34%), but seldom in paired normal colon (0 out of 17) and gastric (1 out of 20, 5%) samples. No correlation between DLEC1 methylation and clinical parameters of gastric cancers was found. Ectopic expression of DLEC1 in silenced HCT116 and MKN45 cells strongly inhibited their clonogenicity. Thus, DLEC1 is a functional tumour suppressor, being frequently silenced by epigenetic mechanism in gastrointestinal tumours.

Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing

Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active.

Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

Nucleolar Dominance and DNA Methylation Directed by Small Interfering RNA

In the December 5th issue of Molecular Cell, Preuss et al. (2008) demonstrated a link between small interfering RNA (siRNA)-directed de novo DNA methylation and rDNA silencing in nucleolar dominance.

Definition of Commonly Deleted Regions and Identification of Candidate Genes of Chromosome 20 in Myeloid Malignancies Using Genome-Wide Single Nucleotide Polymorphism (SNP) Array.

Deletion of the long arm of chromosome 20 (20q) is one of the more common recurring abnormalities associated with myeloid malignancies. The previous studies utilizing FISH and microsatellite PCR attempted to define the commonly deleted regions (CDR) of chromosome 20, but due to the limited resolution of conventional technologies, the precise delineation of such a region was difficult. Among our cohort of 1162 patients with myeloid malignancies, 3% of the patients (34/1162) showed 20q deletion (27 MDS, 2 AML, 5 MDS/MPD). The incidence of 20q deletion as a sole cytogenetic abnormality was 44% (15/34). To decrease the possible size of CDR and better delineate the boundaries of areas of copy-neutral LOH, we analyzed a subcohort of 532 of the patients using high density single nucleotide polymorphism microarray (SNP-A) with 250K and SNP 6.0 including 1.8x106 markers. As a result, we were able to identify chromosome 20q deletion in 21 patients (2 RARS, 3 RCMD, 6 RAEB, 4 MDS-U, 2 AML, 2 MPD, 2 MDS/MPD), while metaphase cytogenetics in the same cohort of patient revealed only 14 cases with 20q deletion. Although the size and breakpoints of the deleted regions varied, they showed considerable overlap. We have defined 2 CDRs on 20q shared by all patients with deletion of chromosome 20q. CDR1 (present 18/21 patients) spanned 2.5Mb and is located between bands 20q11.23 and 20q12 (34,968,632-37,417,814), while CDR2 (present in 21/21 patients) encompassed 1.8Mb within bands 20q13.12 (43,636,105-45,417,399). We found 3 patients (1 RAEB and 2 MDS-U) whose lesions included CDR2 only and without CDR1. Two patients with MDS-U had 20q deletion as a sole abnormality and belong to the low and intermediate-1 risk group, respectively. One RAEB patient who only had CDR2 had duplication of the short arm of chromosome 20 and progressed to AML. As most of the lesions were interstitial, the terminal regions of chromosome 20q and the short arms of chromosome 20 were retained or even duplicated in 8 cases. For RARS and MPD, MDS/MPD patients, there were no patients that have duplicated regions. CDR1 and CDR2 spanned 31 and 33 genes known to be expressed in hematopoietic tissues. We selected 3 candidate genes (retinoblastoma like protein 1), CTNNBL1 (beta-catenine like protein 1) and TP53RK (TP53 regulating kinase) and we did not find any inactivating mutations when the genes were sequenced. Theoretically, deletions result in haploinsufficiency but underexpression may be due to methylation silencing which can result in events similar to a functional knockout if it affects genes in the deleted area. Using 27K methylation arrays, we studied methylation of genes corresponding to CDR1 and CDR2 (10 patients with 20q deletion and 53 patients without 20q deletion). CpG islands that were hypermethylated within these CDR regions relative to controls were studied and there was no hypermethylated CpG site in >25% of patients for CDR1. There were 4 hypermethylated CpG sites (MMP9, SLC12A5, SLC13A3 and CHD22) in >25% of patients for CDR2. It is still unclear if SLC12A5, SLC13A3 and CDH22 are associated with cancer. Among 10 patients with 20q CDR2 deletions, we found 3 patients with hypermethylated CpG sites within MMP9 gene (1 AML, 2 MDS-U). Based on the assumption that methylation can results in deficient gene expression analogous to haploinsufficiency and hypermethylation of genes corresponding to the CDRs in patients without deletion could result in a similar phenotype. Among 53 patients without deletion of 20q, we found hypermethylated MMP9 genes in 42% (22/53) of patients (13 AML, 1 RA, 4 RCMD, 1 RAEB, 1 5q- syndrome, 1 MDS/MPD). Among 10 patients with 20q deletion, there was no evidence of hypermethylation in our candidate genes of RBL1, CTNNBL1 and TP53RK genes. For CTNNBL1 gene, 25% (13/53) of patients without 20q deletion showed hypermethylation (8 AML, 1 RAEB, 3 RCMD, 1 MDS-U). In conclusion, our results suggest that CDR1 and CDR2 regions of chromosome 20 which were delineated by SNP-A may be associated with pathogenesis of myeloid malignancies and candidate genes of RBL1, CTNNBL1, TP53RK and MMP9 need to be further studied.

Mecp2-Null Mice Provide New Neuronal Targets for Rett Syndrome

BackgroundRett syndrome (RTT) is a complex neurological disorder that is one of the most frequent causes of mental retardation in women. A great landmark in research in this field was the discovery of a relationship between the disease and the presence of mutations in the gene that codes for the methyl-CpG binding protein 2 (MeCP2). Currently, MeCP2 is thought to act as a transcriptional repressor that couples DNA methylation and transcriptional silencing. The present study aimed to identify new target genes regulated by Mecp2 in a mouse model of RTT.Methodology/Principal FindingsWe have compared the gene expression profiles of wild type (WT) and Mecp2-null (KO) mice in three regions of the brain (cortex, midbrain, and cerebellum) by using cDNA microarrays. The results obtained were confirmed by quantitative real-time PCR. Subsequent chromatin immunoprecipitation assays revealed seven direct target genes of Mecp2 bound in vivo (Fkbp5, Mobp, Plagl1, Ddc, Mllt2h, Eya2, and S100a9), and three overexpressed genes due to an indirect effect of a lack of Mecp2 (Irak1, Prodh and Dlk1). The regions bound by Mecp2 were always methylated, suggesting the involvement of the methyl-CpG binding domain of the protein in the mechanism of interaction.ConclusionsWe identified new genes that are overexpressed in Mecp2-KO mice and are excellent candidate genes for involvement in various features of the neurological disease. Our results demonstrate new targets of MeCP2 and provide us with a better understanding of the underlying mechanisms of RTT.

Absence of MGMT promoter methylation in endometrial cancer

Objective O 6-methylguanine-DNA methyltransferase (MGMT) acts to repair DNA damaged by alkylation of guanine residues. MGMT promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes [Ogino S, Meyerhardt JA, Kawasaki T, et al. CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma. Virchows Arch 2007;450:529–37; Rodriguez MJ, Acha A, Ruesga MT, Rodriguez C, Rivera JM, Aguirre JM. Loss of expression of DNA repair enzyme MGMT in oral leukoplakia and early oral squamous cell carcinoma. A prognostic tool? Cancer Lett 2007;245:263–8; Ishii T, Murakami J, Notohara K, et al. Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa. Gut 2007;56:13–9]. The loss of MGMT activity and promoter methylation is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas [Weaver KD, Grossman SA, Herman JG. Methylated tumor-specific DNA as a plasma biomarker in patients with glioma. Cancer Invest 2006;24:35–40; Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343:1350–4; Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352:997–1003]. We sought to determine if MGMT promoter methylation plays a role in endometrial cancer. Methods One hundred and twenty primary endometrial cancers were analyzed for MGMT promoter methylation by combined bisulfite restriction analysis (COBRA). The cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. Twenty-one endometrioid and mixed endometrioid ovarian cancers were also analyzed. A subset of the primary tumors was analyzed for MGMT expression by immunohistochemistry. Results No MGMT promoter methylation was seen in the 120 endometrial cancers evaluated or the 6 endometrial cancer cell lines. One of the 21 endometrioid ovarian cancers showed methylation. Immunohistochemistry revealed moderate to high level expression of MGMT in the primary endometrial tumors. Conclusion MGMT promoter methylation is an infrequent event in endometrial cancer. MGMT expression and the ability to repair damaged alkylguanine residues could in part explain the limited response of endometrial tumors to alkylating chemotherapy.

DLC1 tumor suppressor gene inhibits migration and invasion of multiple myeloma cells through RhoA GTPase pathway

DLC1 (deleted in liver cancer 1), a tumor suppressor gene that encodes a RhoGTPase-activating protein, is recurrently downregulated or silenced in various solid tumors and hematological malignancies because of epigenetic modifications or genomic deletion. Here, we identified DLC1 promoter hypermethylation in 43 out of 44 multiple myeloma (MM) cell lines, which resulted in downregulation or silencing of DLC1 in 41 samples. High frequency of tumor-specific methylation and attenuation or silencing of DLC1 expression could serve as an independent diagnostic marker for MM. Combined treatment with demethylating and acetylating agents significantly elevated the expression of DLC1 and suppressed MM cell proliferation. Two cell lines exhibiting complete promoter methylation and the absence of DLC1 expression were transduced by an adenoviral vector containing DLC1 cDNA. In both cell lines, the reexpression of DLC1 inhibited myeloma cell invasion and migration, reduced RhoA activity and resulted in the reorganization of actin cytoskeleton. These results provide the first evidence for the antiproliferative effect of DLC1 in a hematological cancer and implicate RhoA pathway in suppression of MM migration and invasion. Given the myeloma cells sensitivity to the reactivation of DLC1 function, the potential for molecular targeted therapy of DLC1-mediated pathways as well as epigenetic therapies hold prospects.

A Position Effect on the Heritability of Epigenetic Silencing

In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks.

G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription

Methylation of DNA and lysine 9 of histone H3 (H3K9) are well-conserved epigenetic marks for transcriptional silencing. Although H3K9 methylation directs DNA methylation in filamentous fungi and plants, this pathway has not been corroborated in mammals. G9a and GLP/Eu-HMTase1 are two-related mammalian lysine methyltransferases and a G9a/GLP heteromeric complex regulates H3K9 methylation of euchromatin. To elucidate the function of G9a/GLP-mediated H3K9 methylation in the regulation of DNA methylation and transcriptional silencing, we characterized ES cells expressing catalytically inactive mutants of G9a and/or GLP. Interestingly, in ES cells expressing a G9a-mutant/GLP complex that does not rescue global H3K9 methylation, G9a/GLP-target genes remain silent. The CpG sites of the promoter regions of these genes were hypermethylated in such mutant ES cells, but hypomethylated in G9a- or GLP-KO ES cells. Treatment with a DNA methyltransferase inhibitor reactivates these G9a/GLP-target genes in ES cells expressing catalytically inactive G9a/GLP proteins, but not the wild-type proteins. This is the first clear evidence that G9a/GLP suppresses transcription by independently inducing both H3K9 and DNA methylation.

Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line

Epigenetic reprogramming is commonly observed in cancer, and is hypothesized to involve multiple mechanisms, including DNA methylation and Polycomb repressive complexes (PRCs). Here we devise a new experimental and analytical strategy using customized high-density tiling arrays to investigate coordinated patterns of gene expression, DNA methylation, and Polycomb marks which differentiate prostate cancer cells from their normal counterparts. Three major changes in the epigenomic landscape distinguish the two cell types. Developmentally significant genes containing CpG islands which are silenced by PRCs in the normal cells acquire DNA methylation silencing and lose their PRC marks (epigenetic switching). Because these genes are normally silent this switch does not cause de novo repression but might significantly reduce epigenetic plasticity. Two other groups of genes are silenced by either de novo DNA methylation without PRC occupancy (5mC reprogramming) or by de novo PRC occupancy without DNA methylation (PRC reprogramming). Our data suggest that the two silencing mechanisms act in parallel to reprogram the cancer epigenome and that DNA hypermethylation may replace Polycomb-based repression near key regulatory genes, possibly reducing their regulatory plasticity.

Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.

Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer

Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G1 cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (1/5; 20%) and pancreatic (3/19; 15.7%) carcinoma lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53/DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors.

Asbestos exposure predicts cell cycle control gene promoter methylation in pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rapidly fatal tumor with increasing incidence worldwide responsible for many thousands of deaths annually. Although there is a clear link between exposure to asbestos and mesothelioma, and asbestos is known to be both clastogenic and cytotoxic to mesothelial cells, the mechanisms of causation of MPM remain largely unknown. However, there is a rapidly emerging literature that describes inactivation of a diverse array of tumor suppressor genes (TSGs) via promoter DNA CpG methylation in MPM, although the etiology of these alterations remains unclear. We studied the relationships among promoter methylation silencing, asbestos exposure, patient demographics and tumor histology using a directed approach; examining six cell cycle control pathway TSGs in an incident case series of 70 MPMs. Promoter hypermethylation of APC, CCND2, CDKN2A, CDKN2B, HPPBP1 and RASSF1 were assessed. We observed significantly higher lung asbestos body burden if any of these cell cycle genes were methylated (P < 0.02), and there was a significant trend of increasing asbestos body counts as the number of methylated cell cycle pathway genes increased from 0 to 1 to >1 (P < 0.005). This trend of increasing asbestos body count and increasing number of methylated cell cycle pathway genes remained significant (P < 0.05) after controlling for age, gender and tumor histology. These data suggest a novel tumorigenic mechanism of action of asbestos and may contribute to the understanding of precisely how asbestos exposure influences the etiology and clinical course of malignant mesothelioma.