Tackling the methylome: recent methodological advances in genome-wide methylation profiling

DNA methylation of promoter CpG islands is strongly associated with gene silencing and is known as a frequent cause of loss of expression of tumor suppressor genes, as well as other genes involved in tumor formation. DNA methylation of driver genes is very likely outnumbered by the number of methylated passenger genes, though these can be useful as tumor markers. Much of what is known about the importance of DNA methylation in cancer was gained through small- and moderate-scale analysis of gene promoters and tumor samples. A much better understanding of the role of DNA methylation in cancer, either as a marker of disease or as an active driver of tumorigenesis, will likely be gained from genome-wide studies of this modification in normal and malignant cells. This goal has become more attainable with the recent introduction of large-scale genome analysis methodologies and these have been modified to allow for investigation of DNA methylation. Several research groups have been formed to coordinate efforts and apply these methodologies to decipher the methylome of healthy and diseased tissues. In this article we review technological advances in genome-wide methylation profiling

Alterações cromossômicas causadas pela radiação dos monitores de vídeo de computadores

OBJECTIVE: Concerns were raised about the potential damaging effects of electromagnetic field (EMF) radiation emissions of ELF (extremely low frequency) and VLF (very low frequency) computer video display monitors (VDM), it was assessed the frequency of structural chromosome abnormalities and investigated the cell cycle kinetics in individuals occupationally exposed to VDM radiation. METHODS: Chromosome aberrations were investigated in 2,000 first cell cycle metaphases obtained after 48-hour cultures of peripheral blood lymphocytes drawn from 10 individuals occupationally exposed to VDM radiation (group E) and 10 controls (group C). Cell cycle kinetics was studied using the mitotic index (MI) and cellular proliferation index (CPI). RESULTS: Statistical analysis showed significantly higher frequencies of anomalous metaphases (E=5.9%; C=3.7%) and anomaly/cell (E=0.066±0.026; C= 0.040±0.026) in individuals exposed to VDM radiation. The most common cytogenetic alterations seen were chromatid breaks at frequencies of 0.034±0.016 in group E and 0.016±0.015 in group C. There was no significant difference between MI and CPI frequencies in both groups. CONCLUSIONS: The study findings suggest genotoxic effects of EMF emissions revealed by the higher frequency of chromatid breaks in individuals exposed to VDM radiation. However, there is a need of further studies on EMF genetic effects using other investigation methods.OBJETIVO: Em decorrência dos questionamentos sobre o efeito deletério das radiações emitidas pelo campo eletromagnético (CEM) dos tipos ELF (extremely low frequency) e VLF (very low frequency) transmitidas pelos monitores de vídeo dos computadores (CRT), foi avaliada a freqüência de anomalias cromossômicas estruturais e a cinética do ciclo celular em indivíduos expostos por seu trabalho à radiação dos CRT. MÉTODOS: A pesquisa de aberrações cromossômicas foi realizada em 2.000 metáfases de primeira divisão celular obtidas de culturas de 48h de linfócitos de sangue venoso periférico de dez indivíduos expostos ao CRT (grupo E) e de dez controles (grupo C). A cinética do ciclo celular foi pesquisada pelos índices mitótico (IM) e de proliferação celular (IPC). RESULTADOS: A análise estatística evidenciou freqüências significativamente maiores de metáfases com anomalias cromossômicas (E=5,9%; C=3,7%) e anomalias/célula (E=0,066±0,026; C=0,040±0,026) nos indivíduos expostos aos CRTs. As alterações citogenéticas mais comuns foram as quebras cromatídicas, com freqüência de 0,034±0,016 no grupo E e de 0,016±0,015 no grupo C. As freqüências de IM e IPC não apresentaram diferenças significantes entre os grupos avaliados. CONCLUSÕES: Os resultados sugerem um efeito genotóxico do CEM emitido pelos CRTs devido à freqüência mais elevada de quebras cromatídicas, enfatizando a necessidade de haver um número maior de estudos com diferentes técnicas que vise a investigar a ação do CEM sobre o material genético

An Sp1/Sp3 Binding Polymorphism Confers Methylation Protection

Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state

LINE-1 Hypomethylation in Cancer Is Highly Variable and Inversely Correlated with Microsatellite Instability

BACKGROUND: Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer. METHODOLOGY/PRINCIPAL FINDINGS: We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process. CONCLUSION/SIGNIFICANCE: Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer

Epigenomic analysis of aberrantly methylated genes in colorectal cancer identifies genes commonly affected by epigenetic alterations.

Methylation profiling based on bead-chip arrays is an effective method for screening aberrantly methylated genes in CRC. In addition, we identified novel methylated genes that are candidate diagnostic or prognostic markers for CRC

Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray

Objective To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts. Design A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays. Setting University research laboratory. Patient(s) Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom. Intervention(s) None. Main Outcome Measure(s) Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos. Result(s) Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs. Conclusion(s) The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts

Role of DNA methylation in head and neck cancer

Head and neck cancer (HNC) is a heterogenous and complex entity including diverse anatomical sites and a variety of tumor types displaying unique characteristics and different etilogies. Both environmental and genetic factors play a role in the development of the disease, but the underlying mechanism is still far from clear. Previous studies suggest that alterations in the genes acting in cellular signal pathways may contribute to head and neck carcinogenesis. In cancer, DNA methylation patterns display specific aberrations even in the early and precancerous stages and may confer susceptibility to further genetic or epigenetic changes. Silencing of the genes by hypermethylation or induction of oncogenes by promoter hypomethylation are frequent mechanisms in different types of cancer and achieve increasing diagnostic and therapeutic importance since the changes are reversible. Therefore, methylation analysis may provide promising clinical applications, including the development of new biomarkers and prediction of the therapeutic response or prognosis. In this review, we aimed to analyze the available information indicating a role for the epigenetic changes in HNC

Canine Brachycephaly is Associated with a Retrotransposon-Mediated Missplicing of SMOC2

In morphological terms, “form” is used to describe an object’s shape and size. In dogs, facial form is stunningly diverse. Facial retrusion, the proximodistal shortening of the snout and widening of the hard palate is common to brachycephalic dogs and is a welfare concern, as the incidence of respiratory distress and ocular trauma observed in this class of dogs is highly correlated with their skull form. Progress to identify the molecular underpinnings of facial retrusion is limited to association of a missense mutation in BMP3 among small brachycephalic dogs. Here, we used morphometrics of skull isosurfaces derived from 374 pedigree and mixed-breed dogs to dissect the genetics of skull form. Through deconvolution of facial forms, we identified quantitative trait loci that are responsible for canine facial shapes and sizes. Our novel insights include recognition that the FGF4 retrogene insertion, previously associated with appendicular chondrodysplasia, also reduces neurocranium size. Focusing on facial shape, we resolved a quantitative trait locus on canine chromosome 1 to a 188-kb critical interval that encompasses SMOC2. An intronic, transposable element within SMOC2 promotes the utilization of cryptic splice sites, causing its incorporation into transcripts, and drastically reduces SMOC2 gene expression in brachycephalic dogs. SMOC2 disruption affects the facial skeleton in a dose-dependent manner. The size effects of the associated SMOC2 haplotype are profound, accounting for 36% of facial length variation in the dogs we tested. Our data bring new focus to SMOC2 by highlighting its clinical implications in both human and veterinary medicine