The base 5-hydroxymethylcytosine (5hmC) was recently identified as an oxidation product of 5-methylcytosine (5mC) in mammalian DNA. Here, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, we investigated whether levels of 5hmC can distinguish normal tissue from tumor tissue. In squamous cell lung cancers, levels of 5hmdC were depleted substantially with up to 5-fold reduction compared to normal lung tissue. In brain tumors, 5hmdC showed an even more drastic reduction with levels up to >30-fold lower than in normal brain, but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1 (IDH1). Furthermore, immunohistochemical analysis indicated that 5hmC is remarkably depleted in many types of human cancer. Importantly, an inverse relationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. The data therefore suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.
Using a methylated-DNA enrichment technique (methylated CpG island recovery assay, MIRA) in combination with whole-genome tiling arrays, we have characterized by MIRA-chip the entire B cell ''methylome'' of an individual human at 100-bp resolution. We find that at the chromosome level high CpG methylation density is correlated with subtelomeric regions and Giemsa-light bands (R bands). The majority of the most highly methylated regions that could be identified on the tiling arrays were associated with genes. Approximately 10% of all promoters in B cells were found to be methylated, and this methylation correlates with low gene expression. Notably, apparent exceptions to this correlation were the result of transcription from previously unidentified, unmethylated transcription start sites, suggesting that methylation may control alternate promoter usage. Methylation of intragenic (gene body) sequences was found to correlate with increased, not decreased, transcription, and a methylated region near the 3 end was found in approximately 12% of all genes. The majority of broad regions (10 -44 kb) of high methylation were at segmental duplications. Our data provide a valuable resource for the analysis of CpG methylation patterns in a differentiated human cell type and provide new clues regarding the function of mammalian DNA methylation.chromosome structure ͉ DNA methylation ͉ epigenetics ͉ CpG island
Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.
We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type-dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low falsepositive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms. (Cancer Res 2006; 66(16): 7939-47)
De novo methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four HOX gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as SIX, LHX, PAX, DLX, and Engrailed, were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous HOX genes showed that not all paralogues undergo cancerassociated methylation simultaneously. The HOXA cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7-and HOXA9-associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.DNA methylation ͉ HOX genes ͉ chromatin ͉ Polycomb
Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80 -100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.DNA methylation ͉ tiling arrays ͉ CpG islands C hanges in DNA methylation patterns are frequent events in human tumors (1). DNA hypomethylation in cancer tissue was first observed more than two decades ago (2-6) and may be mechanistically linked to tumorigenesis (7). In the 1990s, researchers reported hypermethylation of CpG islands of several known and putative tumor suppressor genes and other genes involved in important genome defense pathways, such as DNA repair (1,(8)(9)(10)(11)(12). Today, there are many reports that have documented methylation of CpG islands associated with a large number of different genes, including almost every type of human cancer. In lung cancer, several CpG islands known to be methylated include those associated with CDKN2A, RASSF1A, RARbeta, MGMT, GSTP1, CDH13, APC, DAPK, TIMP3,. The methylation frequency (i.e., the percentage of tumors analyzed that carry methylated alleles) ranges from Ͻ10% to Ϸ80%, but these numbers differ widely depending on the tumor histology, the study population, and/or the methodology used to assess methylation. Detection of methylated CpG islands in easily accessible biological materials such as serum or sputum has the potential to be useful for the early diagnosis of lung cancer and other malignancies (18)(19)(20).Repetitive DNA elements, such as short and long interspersed nuclear elements (SINEs and LINEs, respectively) and simple repeat sequences, are often found hypomethylated in tumors (21-26). Although it seems clear that methylation-induced silencing of tumor suppressor genes can be an important event in tumorigenesis, the magnitude, exact sequence specificity, and biological significance of tumor-associated DNA hypomethylation is much less understood (21, 26). In particular, the extent and sequence context of single-copy gene and general genome hy...
DNA CpG methylation can cooperate with histone H3 lysine 9 (H3-K9) methylation in heterochromatin formation and gene silencing. Trimethylation of H3-K9 by the recently identified euchromatic histone methyltransferase SETDB1/ESET may be responsible for transcriptional repression of certain promoters. Here, we show that SETDB1 associates with endogenous DNA methyltransferase activity. SETDB1 interacts with the de novo DNA methyltransferases DNMT3A and DNMT3B but not with the maintenance methyltransferase DNMT1. The interaction of SETDB1 with DNMT3A was further characterized and confirmed by in vivo and in vitro interaction studies. A direct interaction of the two proteins occurs through the N terminus of SETDB1 and the plant homeodomain of DNMT3A. Co-expression of SETDB1 and DNMT3A was essential for repression of reporter gene expression in a Gal4-based tethering assay and resulted in their recruitment to the artificial promoter. We further demonstrate that the CpG-methylated promoters of the endogenous p53BP2 gene in HeLa cells and the RASSF1A gene in MDA-MB-231 cells are simultaneously occupied by both SETDB1 and DNMT3A proteins, which provides evidence for SETDB1 being at least partly responsible for H3-K9 trimethylation at the promoter of RASSF1A, a gene frequently silenced in human cancers. In summary, our data demonstrate the direct physical interaction and functional connection between the H3-K9 trimethylase SETDB1 and the DNA methyltransferase DNMT3A and thus contribute to a better understanding of the complexity of the self-reinforcing heterochromatin machinery operating at silenced promoters.Epigenetic gene regulation is a process that can generate heritable marks on DNA and histone N-terminal tails, which are crucial to maintain the stable patterns of gene expression. Methylation of cytosine within the context of CpG dinucleotides and histone H3 lysine 9 (H3-K9) 2 are two important epigenetic modifications, high levels of which are characteristic of transcriptionally silenced gene promoters and constitutive heterochromatin. Mammalian CpG methylation is carried out by three active DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B. DNMT1 mediates replication-coupled maintenance of DNA methylation patterns, whereas DNMT3A and DNMT3B are considered to be de novo DNA methylases, which are critical in the dynamic DNA methylation process during embryogenesis and pathogenesis (1-3).Recently, additional involvements of DNMT3A and DNMT3B in the maintenance of DNA methylation patterns have also been reported (4, 5). Unlike DNA methylation that commonly leads to permanent gene silencing, histone modifications, such as acetylation, phosphorylation, and methylation, exert diversified and presumably more reversible effects on gene transcriptional regulation. Histone methylation occurs on both arginines and lysines, such as arginine 17 and lysine 9 of H3 that mark opposite transcription states (6). Histone H3-K9 methylation is catalyzed by members of the SET (SuVar3-9, enhancer of Zeste, Trithorax) domain-conta...
BackgroundInsulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression.Methodology/Principal FindingsGenomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro.Conclusions/SignificanceOur findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.
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