5-Methyl-2''-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.     

The role of epigenetic alterations in pancreatic cancer

The past several years have witnessed an explosive increase in our knowledge about epigenetic features in human cancers. It has become apparent that pancreatic cancer is an epigenetic disease, as it is a genetic disease, characterized by widespread and profound alterations in DNA methylation. The introduction of genome-wide screening techniques has accelerated the discovery of a growing list of genes with abnormal methylation patterns in pancreatic cancer, and some of these epigenetic events play a role in the neoplastic process. The detection and quantification of DNA methylation alterations in pancreatic juice is likely a promising tool for the diagnosis of pancreatic cancer. The potential reversibility of epigenetic changes in genes involved in tumor progression makes them attractive therapeutic targets, but the efficacy of epigenetic therapies in pancreatic cancer, such as the use of DNA methylation inhibitors, remains undetermined. In this review, we briefly summarize recent research findings in the field of pancreatic cancer epigenetics and discuss their biological and clinical implications.     

A unique configuration of genome-wide DNA methylation patterns in the testis

In the mammalian lifecycle, the two periods of genome-wide epigenetic reprogramming are in the early embryo, when somatic patterns are set, and during germ cell development. Although some differences between the reprogrammed states of somatic and germ cells have been reported, overall patterns of genomic methylation are considered to be similar. Using restriction landmark genomic scanning to examine approximately 2,600 loci distributed randomly throughout the genome, we find that the methylation status of testicular DNA is highly distinct, displaying eightfold the number of hypomethylated loci relative to somatic tissues. Identification and analysis of >300 loci show that these regions are generally located within nonrepetitive sequences that are away from CpG islands and 5' regions of genes. We show that a contributing factor for these differences is that the methylation state of non-CpG-island DNA is correlated with the regional level of GC content within chromosomes and that this relationship is inverted between the testis and somatic tissues. We also show that in Dnmt3L-deficient mice, which exhibit infertility associated with abnormal chromosomal structures in germ cells, this unique testicular DNA methylation pattern is not established. These special properties of testicular DNA point to a broad, distinct epigenetic state that may be involved in maintaining a unique chromosomal structure in male germ cells.     

DNA methylation in liver diseases

Recently, growing evidences show that the combination of epigenetic and genetic abnormalities contribute together to the development of liver diseases. DNA methylation is a very important epigenetic mechanism in human beings. It refers to addition of the methyl groups to DNA and mainly occurs at cytosine adjacent to guanine. DNA methylation is prevalent across human genome and is essential for the normal human development, while its dysfunction is associated with many human diseases. A deep understanding of DNA methylation may provide us deep insight into the origination of liver diseases. Also, it may provide us new tools for diseases diagnosis and prognosis prediction. This review summarized recent progress of DNA methylation study and provided an overview of DNA methylation and liver diseases. Meanwhile, the association between DNA methylation and liver diseases including hepatocellular carcinoma, liver fibrosis, nonalcoholic steatohepatitis and liver failure were extensively discussed. Finally, we discussed the potential of DNA methylationtherapeutics for liver diseases and the value of DNA methylation as biomarkers for liver diseases diagnosis and prognosis prediction. This review aimed to provide the emerging DNA methylation information about liver diseases. It might provide essential information for managing and care of these patients.     

Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats

There is a concern that increased paternal age may be associated with altered fertility and an increased incidence of birth defects in man. In previous studies of aged male rats, we have found abnormalities in the fertility and in the embryos sired by older males. Aging in mammals is associated with alterations in the content and patterns of DNA methylation in somatic cells; however, little is known in regard to germ cells. A systematic search for global and gene-specific alterations of DNA methylation in germ cells and liver of male rats was done. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, has revealed a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. In contrast, all single copy CpG island sequences in spermatozoa and in liver remain unaltered with age. We further demonstrate that a large proportion of rat ribosomal DNA is normally methylated and that regional and site-specific differences exist in the patterns of methylation between spermatozoa and liver. We conclude that patterns of ribosomal DNA methylation in spermatozoa are vulnerable to the same age-dependent alterations that we observe in normal aging liver. Failure to maintain normal DNA methylation patterns in male germ cells could be one of the mechanisms underlying age-related abnormalities in fertility and progeny outcome.     

Abnormal regional hypermethylation in cancer cells.

Let me summarize by reviewing a model which is meant to raise as many questions as it answers (Fig. 2). What I have discussed today are data suggesting that during progression of solid tumors, like colon cancer, an increased cellular DNA methylating capacity characterizes the initial stages of multi-clonal hyperplasia. Despite this increase, the altered pattern of DNA methylation which subsequently emerges is largely manifest by a widespread hypomethylation of DNA. However, on a more regional basis, areas of hypermethylation appear which can affect strategic areas such as normally unmethylated CpG islands. These shifted DNA methylation patterns have the capacity to both follow, or cause, chromatin changes that can both directly silence genes critical for normal cell maturation--and/or participate in the structural chromosome changes which constitute genetic instability during tumor progression (Fig. 2). I suggest that one must view these changes as an interchangeable cycle of events during tumor progression. The chromatin changes and abnormal methylation patterns can drive one another with increasingly deleterious effects as the malignant phenotype emerges (reviewed in Baylin, 1991). What are the molecular events that would initiate the above dynamics? A working construct model is shown in Fig. 3. As discussed for the normal adult cell, there is a delicate balance between the strategic location of DNA MTase, regulation of this enzyme, and rate of DNA synthesis at replication forks (top panel, Fig. 3). In pre-neoplastic and cancer cells, perhaps failure of cells to exit the cell cycle and halt DNA replication, facilitates some sort of pressure to increase cellular DNA methyltransferase activity (bottom panel, Fig. 3). This increase may involve loss of feedback inhibition of the enzyme during the post DNA replication phase. There are also probable structural alterations in the nucleus which may alter the geographic relationship between the DNA replication fork and DNA MTase. In consequence, many DNA areas that should be getting methylated do not, and novel areas of methylation also arise. This cycle of events leads to the imbalance of DNA methylation that I have talked about. Future investigations of these possibilities, and of their specific consequences for alterations of gene expression and chromosome structure, may reveal a key molecular step underlying virtually all stages of tumor progression.     

Reading the unique DNA methylation landscape of the brain: Non-CpG methylation,hydroxymethylation, and MeCP2

DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.     

Expression of chorionic gonadotropin alpha- and beta-genes in normal and neoplastic human tissues: relationship to deoxyribonucleic acid structure

We have investigated whether the expression of hCG genes can be attributed to changes in the structure of the alpha- and beta hCG genes, such as rearrangements, duplications, or methylation patterns. Various tissues and cell lines were studied: two term placentae, three trophoblastic tumor cell lines, two tumor cell lines ectopically producing alpha-subunit, normal cells not producing hCG or subunits, and a nonproducing malignancy. Gene structure was explored by restriction enzyme analysis and Southern blotting of DNA, using as probes 32P-labeled plasmids containing alpha- and beta hCG cDNAs. Similarly, methylation was evaluated using the restriction enzymes Msp I, Hpa II, and Hha I, each sensitive to a different pattern of cytosine methylation. No structural changes were observed in alpha- and beta hCG genes, although certain polymorphisms were observed. Analysis of methylation patterns revealed variation of the methylated cytosines; however, no clear correlation was seen between overall methylation or a specific pattern of methylation of these genes and their expression. Although specific methylated nucleotides of regulatory importance may not have been detected by our methods, we can still conclude that neither DNA structural alterations nor patterns of cytosine methylation appear to be major determinants of hCG expression.     

Could Sirt1-mediated epigenetic effects contribute to the longevity response to dietary restriction and be mimicked by other dietary interventions?

Dietary restriction (DR) increases lifespan in a range of evolutionarily distinct species. The polyphenol resveratrol may be a dietary mimetic of some effects of DR. The pivotal role of the mammalian histone deacetylase (HDAC) Sirt1, and its homologue in other organisms, in mediating the effects of both DR and resveratrol on lifespan/ageing suggests it may be the common conduit through which these dietary interventions influence ageing. We propose the novel hypothesis that effects of DR relevant to lifespan extension include maintenance of DNA methylation patterns through Sirt1-mediated epigenetic effects, and proffer the view that dietary components, including resveratrol, may mimic these actions.     

Strand-biased DNA methylation associated with centromeric regions in Arabidopsis

The Arabidopsis genome project assembled 15 megabases of heterochromatic sequence, facilitating investigations of heterochromatin assembly, maintenance, and structure. In many species, large quantities of methylcytosine decorate heterochromatin; these modifications are typically maintained by methyltransferases that recognize newly replicated hemimethylated DNA. We assessed the extent and patterns of Arabidopsis heterochromatin methylation by amplifying and sequencing genomic DNA treated with bisulfite, which converts cytosine, but not methylcytosine, to uracil. This survey revealed unexpected asymmetries in methylation patterns, with one helix strand often exhibiting higher levels of methylation. We confirmed these observations both by immunoprecipitating methylated DNA strands and by restriction enzyme digestion of amplified, bisulfite-treated DNA. We also developed a primer-extension assay that can monitor the methylation status of an entire chromosome, demonstrating that strand-specific methylation occurs predominantly in the centromeric regions. Conventional models for methylation maintenance do not explain these unusual patterns; instead, new models that allow for strand specificity are required. The abundance of Arabidopsis strand-specific modifications points to their importance, perhaps as epigenetic signals that mark the heterochromatic regions that confer centromere activity.     

The emerging roles of DNA methylation in the clinical management of prostate cancer

Aberrant DNA methylation is one of the hallmarks of carcinogenesis and has been recognized in cancer cells for more than 20 years. The role of DNA methylation in malignant transformation of the prostate has been intensely studied, from its contribution to the early stages of tumour development to the advanced stages of androgen independence. The most significant advances have involved the discovery of numerous targets such as GSTP1, Ras-association domain family 1A (RASSF1A) and retinoic acid receptor beta2 (RARbeta2) that become inactivated through promoter hypermethylation during the course of disease initiation and progression. This has provided the basis for translational research into methylation biomarkers for early detection and prognosis of prostate cancer. Investigations into the causes of these methylation events have yielded little definitive data. Aberrant hypomethylation and how it impacts upon prostate cancer has been less well studied. Herein we discuss the major developments in the fields of prostate cancer and DNA methylation, and how this epigenetic modification can be harnessed to address some of the key issues impeding the successful clinical management of prostate cancer.     

Epigenetic control of vasopressin expression is maintained by steroid hormones in the adult male rat brain

Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the socially relevant peptide vasopressin (AVP) within the bed nucleus of the stria terminalis (BST) of adult brain is dependent upon testosterone exposure. Castration dramatically reduces and testosterone replacement restores AVP expression within the BST. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that AVP expression in the adult brain is maintained through sustained epigenetic modifications of the AVP gene promoter. We find that castration of adult male rats resulted in decreased AVP mRNA expression and increased methylation of specific CpG sites within the AVP promoter in the BST. Similarly, castration significantly increased estrogen receptor α (ERα) mRNA expression and decreased ERα promoter methylation within the BST. These changes were prevented by testosterone replacement. This suggests that the DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated or demethylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the homeostatic regulation of behaviorally relevant systems.     

Analysis of DNA modifications in aging research

As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through next-generation sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-the-art for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.     

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.     

CG gene body DNA methylation changes and evolution of duplicated genes in cassava

DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.DNA methylation plays an important role in the regulation of the expression of genes and the maintenance of transposable element (TE) silencing. In contrast to animals, in which methylation is often restricted to the CG context, plants exhibit robust methylation in every possible context CG, CHG (H is A, T, or C), and CHH. Previous research has identified different pathways responsible for the maintenance and establishment of DNA methylation patterns. In Arabidopsis thaliana, METHYLTRANSFERASE1 (MET1), a homolog of mammalian Dnmt1, mainly maintains methylation at the CG context, whereas CHROMOMETHYLASE3 (CMT3) mainly maintains CHG methylation. DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and CHROMOMETHYLASE2 (CMT2) maintain CHH methylation in the chromosome arms and pericentromeric regions, respectively (1–3). On the other hand, establishment of DNA methylation is performed by DRM2 through a complex pathway termed RNA-directed DNA methylation (RdDM) (4).To date, the majority of our knowledge about DNA methylation is derived from the model plant Arabidopsis. These studies have allowed the identification of different components involved in different methylation pathways, the genome-wide identification of methylation patterns, and the study of effects of DNA methylation on gene expression. The knowledge acquired from Arabidopsis can now be used as the basis for investigations of methylation in agronomically important plants. However, thus far very few crop species have been subjected to detailed DNA methylation studies (5). Cassava (Manihot esculenta) is cultivated for its starch-rich tuberous roots and is one of the world’s most important staple crops, especially in tropical America, Africa, and Asia (6). Cassava is a source of carbohydrates for nearly a billion people, but it is especially important for a large portion of Africa, where it serves as a subsistence crop because of its ability to tolerate drought and grow on poor soils, conditions unsuitable for rice and maize (6, 7). The genome sequence of cassava has been described recently with an estimated genome size of roughly 760 million base pairs (7). We have used bisulfite sequencing (BS-seq) to examine DNA methylation in cassava at single-base pair resolution. Broadly, the pattern of DNA methylation of both protein-coding genes and TEs is similar to other plants, although DNA methylation levels in cassava are higher than those in Arabidopsis. LTR retrotransposons, such as GYPSY and COPIA, tend to be more heavily methylated than other TEs. Interestingly, differentially expressed gene pairs derived from the last genome duplication tend to show differential gene body methylation, with the highly expressed paralogs displaying significantly higher gene body methylation. We also find that the most differentially gene body-methylated paralogs have distinct biological functions compared with genes that have maintained similar gene body methylation patterns.     

DNA methylation patterns of the gamma delta beta-globin genes in human fetal and adult erythroid tissues.

An investigation of the correlation between the gamma----beta-globin switch and DNA methylation was carried out. The restriction patterns obtained with methylation-sensitive and -insensitive enzymes indicated hypomethylation in the promoter region of the gamma-globin genes in fetal liver DNA but high methylation of the same region in all other samples (except in the presence of an elevated erythroblast count or leukemia). All samples appeared to be partially hypomethylated at the 5' end of the delta-globin gene and hypomethylated at the 3' region of the beta-globin gene. Although consistent with a role for DNA methylation in globin gene regulation, the results also suggest that other factors besides methylation may be required for regulation of the level of expression, and switching of the globin genes.