DNA motifs associated with aberrant CpG island methylation

Epigenetic silencing involving the aberrant methylation of promoter region CpG islands is widely recognized as a tumor suppressor silencing mechanism in cancer. However, the molecular pathways underlying aberrant DNA methylation remain elusive. Recently we showed that, on a genome-wide level, CpG island loci differ in their intrinsic susceptibility to aberrant methylation and that this susceptibility can be predicted based on underlying sequence context. These data suggest that there are sequence/structural features that contribute to the protection from or susceptibility to aberrant methylation. Here we use motif elicitation coupled with classification techniques to identify DNA sequence motifs that selectively define methylation-prone or methylation-resistant CpG islands. Motifs common to 28 methylation-prone or 47 methylation-resistant CpG island-containing genomic fragments were determined using the MEME and MAST algorithms (). The five most discriminatory motifs derived from methylation-prone sequences were found to be associated with CpG islands in general and were nonrandomly distributed throughout the genome. In contrast, the eight most discriminatory motifs derived from the methylation-resistant CpG islands were randomly distributed throughout the genome. Interestingly, this latter group tended to associate with Alu and other repetitive sequences. Used together, the frequency of occurrence of these motifs successfully discriminated methylation-prone and methylation-resistant CpG island groups with an accuracy of 87% after 10-fold cross-validation. The motifs identified here are candidate methylation-targeting or methylation-protection DNA sequences.     

A human CpG island randomly inserted into a plant genome is protected from methylation

In vertebrate genomes the dinucleotide CpG is heavily methylated, except in CpG islands, which are normally unmethylated. It is not clear why the CpG islands are such poor substrates for DNA methyltransferase. Plant genomes display methylation, but otherwise the genomes of plants and animals represent two very divergent evolutionary lines. To gain a further understanding of the resistance of CpG islands to methylation, we introduced a human CpG island from the proteasome-like subunit I gene into the genome of the plant Arabidopsis thaliana. Our results show that prevention of methylation is an intrinsic property of CpG islands, recognized even if a human CpG island is transferred to a plant genome. Two different parts of the human CpG island – the promoter region/ first exon and exon2–4 – both displayed resistance against methylation, but the promoter/ exon1 construct seemed to be most resistant. In contrast, certain sites in a plant CpG-rich region used as a control transgene were always methylated. The frequency of silencing of the adjacent nptII (Km^R) gene in the human CpG constructs was lower than observed for the plant CpG-rich region. These results have implications for understanding DNA methylation, and for construction of vectors that will reduce transgene silencing.     

CpG island methylation pattern in different human tissues and its correlation with gene expression

The study on DNA methylation pattern in different human tissues attracts increasing interest nowadays, but a systematic analysis of CpG island methylation pattern between both somatic tissues and gametocyte is still lacking. In this work, we analyzed the CpG island methylation data of sperm and other 11 somatic tissues from Human Epigenome Project, and found that the CpG island methylation profiles are highly correlated between somatic tissues, while the methylation profile in sperm is quite distinct. Furthermore, we observed that in the six tissues investigated, there is no obvious correlation between the methylation level of promoter CpG islands and corresponding gene expression across different tissues.     

Distribution of DNA methylation,CpGs, and CpG islands in human isochores

DNA methylation is a major epigenetic modification of the genome that affects basic biological functions, such as gene expression and cell development. We used the human genome sequences and the DNA methylation data that are available in order to establish a map of the levels of GC and methylation in isochores. We also looked for the correlations that hold between GC levels and the distribution of the (1) dinucleotide CpG, (2) ratio 5mC/CpG, and (3) CpG islands. Our results show that methylation levels, CpG frequencies, and the density of CpG islands are positively correlated with the GC level of isochores. In contrast, the correlation between the 5mC/CpG ratio and GC is a negative one because the increase in methylation lags behind that of CpG, to reach a plateau in the GC-richest, gene-richest isochore families H2 and H3. In conclusion, there are more CpG targets that remain unmethylated in the GC-richest, gene-richest isochores in comparison with the other isochores. This conclusion supports the idea that the widespread methylation under consideration here has a general inhibitory effect on gene expression.     

Confluence-induced alterations in CpG island methylation in cultured normal human fibroblasts.

Growth constraint of bacterial and human cells has been shown to trigger genetic mutation. We questioned whether growth constraint might also trigger epigenetic mutation in the form of CpG island methylation. Logarithmically growing normal human fibro-blasts (NHF) displayed little (0-15%) CpG methylation in select regions of three CpG islands [estrogen receptor (ER), E-cadherin (ECAD) and O (6)-methylguanine-DNA methyltransferase (MGMT)] examined. NHF grown to and left at confluence for 2-21 days showed little (<10%) CpG methylation in the ER and ECAD CpG islands. These confluent, growth-arrested cells, however, displayed extensive ( approximately 50%) methylation of the MGMT CpG island. CpG methylation in the MGMT CpG island was not associated with cellular senescence. The methylation was, however, heritable, but not permanent, as the level of CpG methylation in the MGMT CpG island of cells 4 population doublings following replating after confluence were no different from those in confluent cultures, but returned to levels noted in logarithmically growing cells by 10 population doublings following replating. These results suggest that growth constraint can trigger transient epigenetic change even in normal non-senescent human cells.     

SFRP1 CpG island methylation locus is associated with renal cell cancer susceptibility and disease recurrence

Loss of the secreted Fzd-related protein 1 (SFRP1) and concurrent alteration of the SFRP1/WNT pathway are frequently observed in human cancers such as in renal cell cancer (RCC). Whether methylation of a SFRP1 CpG island locus in normal human solid tissues is associated with increased tissue specific cancer risk has not been determined to date. Here we measure the cancer risk attributable to SFRP1 DNA methylation in renal tissue. Pyrosequencing of bisulfite treated DNA was used for a case-control study including 120 normal-appearing renal tissues of autopsy specimens and 72 normal-appearing tissues obtained from tumor adjacent areas, and a cross sectional study of 96 RCCs. Association of methylation with demographic risk factor age, clinicopathological parameters and course of patients was investigated. We show significant hypermethylation of a SFRP1 CpG island locus in normal-appearing renal tissues from RCC patients compared with normal-appearing autopsy kidney tissues. Inter quartile analysis revealed a 6-, 13- and 11-fold increased cancer risk for the second, third and fourth quartiles of methylation in the age matched subgroup of tissues (p = 0.001, p = 1.3E-6, p = 6.9E-6). Methylation in autopsy tissues increased with age and methylation in tumors was an independent predictor of recurrence free survival. SFRP1 DNA methylation, accumulates with age in normal-appearing kidney tissues and is associated with increased renal cancer risk, suggesting this CGI sub region as an epigenetic susceptibility locus for RCC. Our data underline the need to further analyze the tissue specific risks conferred by methylated loci for the development of human cancers.     

Classification of DNA methylation patterns in tumor cell genomes using a CpG island microarray

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample's response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.     

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.     

Polymorphisms of DNA repair and xenobiotic genes predispose to CpG island methylation in non-neoplastic gastric mucosa

Background: Genetic factors, related to DNA repair or xenobiotic pathways might confer different degrees of susceptibility to gastric carcinogenesis. CpG island hyper methylation (CIHM) is a major event in gastric carcinogenesis. We evaluated the association between XRCC1, GSTP1, GSTT1 and GSTM1 polymorphisms with CIHM status in non‐neoplastic gastric mucosa. Methods: XRCC1 Arg399Gln, and Arg194Trp, GSTP1 Ile104Val, and GSTT1, GSTM1 null polymorphisms were genotyped in 415 cancer free subjects, in relation to four candidate CpG (p14, p16, DAP‐kinase and CDH1) loci, assessed by Methylation‐Specific‐Polymerase Chain Reaction (MSP). CIHM high was defined as two or more CpG islands methylated. Results: Significant association between XRCC1 codon 399 Gln/Gln genotype and reduced susceptibility to CIHM of DAP‐kinase (adjusted OR = 0.30, 95%CI = 0.13–0.71, p = .0055) and CIHM high (OR = 0.42, 95%CI = 0.19–0.97, p = .04). XRCC1 codon 399 Gin/Gln genotype also presented lower number of CIHM when compared with both Arg/Gln, and Arg/Arg + Arg/Gln genotypes (p = .02, .046, respectively) When subjects were divided according to age (>50 and <50), an association was found between GSTM1 null genotype and increased susceptibility to CIHM high in the 50 years and older generations (OR = 1.63, 95%CI = 1.01–2.62, p = .045). Conclusion: XRCC1 codon 399 Gln/Gln genotype is associated with reduced susceptibility to CIHM especially DAP‐kinase. GSTM1 null genotype may increase the susceptibility to CIHM especially in older patients. Genetic factors, related to DNA repair or xenobiotic pathways may have a role in CIHM‐related gastric carcinogenesis.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.