Epigenetics: differential DNA methylation in mammalian somatic tissues

Epigenetics refers to heritable phenotypic alterations in the absence of DNA sequence changes, and DNA methylation is one of the extensively studied epigenetic alterations. DNA methylation is an evolutionally conserved mechanism to regulate gene expression in mammals. Because DNA methylation is preserved during DNA replication it can be inherited. Thus, DNA methylation could be a major mechanism by which to produce semi-stable changes in gene expression in somatic tissues. Although it remains controversial whether germ-line DNA methylation in mammalian genomes is stably heritable, frequent tissue-specific and disease-specific de novo methylation events are observed during somatic cell development/differentiation. In this minireview, we discuss the use of restriction landmark genomic scanning, together with in silico analysis, to identify differentially methylated regions in the mammalian genome. We then present a rough overview of quantitative DNA methylation patterns at 4600 NotI sites and more than 150 differentially methylated regions in several C57BL/6J mouse tissues. Comparative analysis between mice and humans suggests that some, but not all, tissue-specific differentially methylated regions are conserved. A deeper understanding of cell-type-specific differences in DNA methylation might lead to a better illustration of the mechanisms behind tissue-specific differentiation in mammals.     

Cleavage of a model DNA replication fork by a methyl-specific endonuclease

Epigenetic DNA methylation is involved in many biological processes. An epigenetic status can be altered by gain or loss of a DNA methyltransferase gene or its activity. Repair of DNA damage can also remove DNA methylation. In response to such alterations, DNA endonucleases that sense DNA methylation can act and may cause cell death. Here, we explored the possibility that McrBC, a methylation-dependent DNase of Escherichia coli, cleaves DNA at a replication fork. First, we found that in vivo restriction by McrBC of bacteriophage carrying a foreign DNA methyltransferase gene is increased in the absence of homologous recombination. This suggests that some cleavage events are repaired by recombination and must take place during or after replication. Next, we demonstrated that the enzyme can cleave a model DNA replication fork in vitro. Cleavage of a fork required methylation on both arms and removed one, the other or both of the arms. Most cleavage events removed the methylated sites from the fork. This result suggests that acquisition of even rarely occurring modification patterns will be recognized and rejected efficiently by modification-dependent restriction systems that recognize two sites. This process might serve to maintain an epigenetic status along the genome through programmed cell death.     

In vitro methylation of DNA with Hpa II methylase.

The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine. The enzyme will methylate this sequence if both DNA strands are unmethylated or if only one strand is unmethylated. Conditions have been developed for producing fully methylated DNA from various sources. In vitro methylation of this site protects the DNA against digestion by the restriction enzyme Hpa II as well as the enzyme Sma I which recognizes the hexanucleotide sequence CCCGGG. These properties make this enzyme a valuable tool for analyzing methylation in eukaryotic DNA where the sequence CCGG is highly methylated. The activity of this methylase on such DNA indicates the degree of undermethylation of the CCGG sequence. Several examples show that this technique can be used to detect small changes in the methylation state of eukaryotic DNA.     

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.     

Methylation analysis by DNA immunoprecipitation

DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)‐based methods to evaluate methylation status of DNA have been instrumental in such genome‐wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome‐wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed. J. Cell. Physiol. 222: 522–531, 2010. © 2009 Wiley‐Liss, Inc.     

A novel sperm-specific hypomethylation sequence is a demethylation hotspot in human hepatocellular carcinomas.

Certain human DNA regions are strikingly undermethylated at CpG sites in sperm compared to adult somatic tissues. These sperm-specific hypomethylation sequences are thought to function early in embryogenesis or gametogenesis. By using the restriction landmark genomic scanning (RLGS) cloning method, we have isolated a novel sperm-specific hypomethylation sequence, the status of which changes during spermatogenesis, embryonal growth and differentiation. This sequence is a part of a new 'NotI repeat' consisting of a 1.4 kb repetitive unit sequence named DE-1. The sequence is GC-rich and has high homology to a CpG DNA clone that was isolated by a methyl CpG protein binding column, indicating that it was normally highly methylated. We investigated the methylation status of this sequence. In the normal genome the sequence was methylated, but in the human hepatocellular carcinoma (HCC) genome, the target sequence was demethylated at the cytosine residue of the CpG dinucleotides with high frequency (75% in the previous study). These data suggest that this regional DNA hypomethylation may play a role in both cell differentiation and hepatocarcinogenesis.     

Immunological detection of changes in genomic DNA methylation during early zebrafish development.

DNA methylation reprogramming, the erasure of DNA methylation patterns shortly after fertilization and their reestablishment during subsequent early development, is essential for proper mammalian embryogenesis. In contrast, the importance of this process in the development of non-mammalian vertebrates such as fish is less clear. Indeed, whether or not any widespread changes in DNA methylation occur at all during cleavage and blastula stages of fish in a fashion similar to that shown in mammals has remained controversial. Here we have addressed this issue by applying the techniques of Southwestern immunoblotting and immunohistochemistry with an anti-5-methylcytosine antibody to the examination of DNA methylation in early zebrafish embryos. These techniques have recently been utilized to demonstrate that development-specific changes in genomic DNA methylation also occur in Drosophila melanogaster and Dictyostelium discoideum, both organisms for which DNA methylation was previously not thought to occur. Our data demonstrate that genome-wide changes in DNA methylation occur during early zebrafish development. Although zebrafish sperm DNA is strongly methylated, the zebrafish genome is not detectably methylated through cleavage and early blastula stages but is heavily remethylated in blastula and early gastrula stages.     

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.     

Recent Retrotransposon Insertions Are Methylated and Phylogenetically Clustered in Japonica Rice (Oryza sativa spp. japonica)

In plants, the genome of the host responds to the amplification of transposable elements (TEs) with DNA methylation. However, neither the factors involved in TE methylation nor the dynamics of the host-TE interaction are well resolved. Here, we identify 5,522 long terminal repeat retrotransposons (LTR-RT) in the genome of Oryza sativa ssp. japonica and then assess methylation for individual elements. Our analyses uncover three strong trends: long LTR-RTs are more highly methylated, the insertion times of LTR-RTs are negatively correlated with methylation, and young LTR-RTs tend to be closer to genes than older insertions. Additionally, a phylogenetic examination of the gypsy-like LTR-RT superfamily revealed that methylation is phylogenetically correlated. Given these observations, we present a model suggesting that the phylogenetic correlation among related LTR-RTs is a primary mechanism driving methylation. In this model, bursts of transposition produce new elements with high sequence similarity. The host machinery identifies proliferating elements as well as closely related LTR-RTs through cross-homology. In addition, our data are consistent with previous hypotheses that methylated LTR-RT elements are removed preferentially from regions near genes, explaining some of the observed age distribution.     

High-throughput assay of DNA methylation based on methylation-specific primer and SAGE

Mapping of genomic DNA methylation is a dispensable part of functional genome. We have developed a novel method based on methylation-specific primer and serial analysis of gene expression, called MSP-SAGE, with potential of high-throughput quantification of genomic DNA methylation. We used a 6-mer methylation-specific primer to extend the methylated CpG sequences other than non-methylated CpG sequences. The 17 bp tags contained methylated CpG sequence, which were obtained from extended methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing. We can identify the locations of methylation according to the sequences of tags and quantify the methylation status from the frequency of the tags. MSP-SAGE has a good linearity in a broad methylation range from 5% to 100% with good accuracy and high precision. The proof-of-principle study shows that MSP-SAGE is a reliable high-throughput assay for quantification of DNA methylation.     

Genic DNA methylation changes during in vitro organogenesis: organ specificity and conservation between parental lines of epialleles

During differentiation, in vitro organogenesis calls for the adjustment of the gene expression program toward a new fate. The role of epigenetic mechanisms including DNA methylation is suggested but little is known about the loci affected by DNA methylation changes, particularly in agronomic plants for witch in vitro technologies are useful such as sugar beet. Here, three pairs of organogenic and non-organogenic in vitro cell lines originating from different sugar beet (Beta vulgaris altissima) cultivars were used to assess the dynamics of DNA methylation at the global or genic levels during shoot or root regeneration. The restriction landmark genome scanning for methylation approach was applied to provide a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence that is particularly adapted for studies on crop plants without a fully sequenced genome. The cloned sequences had putative roles in cell proliferation, differentiation or unknown functions and displayed organ-specific DNA polymorphism for methylation and changes in expression during in vitro organogenesis. Among them, a potential ubiquitin extension protein 6 (UBI6) was shown, in different cultivars, to exhibit repeatable variations of DNA methylation and gene expression during shoot regeneration. In addition, abnormal development and callogenesis were observed in a T-DNA insertion mutant (ubi6) for a homologous sequence in Arabidopsis. Our data showed that DNA methylation is changed in an organ-specific way for genes exhibiting variations of expression and playing potential role during organogenesis. These epialleles could be conserved between parental lines opening perspectives for molecular markers.     

DNA Sequence Explains Seemingly Disordered Methylation Levels in Partially Methylated Domains of Mammalian Genomes

For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%–40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R²) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.     

Clustering and methylation of repeated DNA: persistence in avian development and evolution.

In the chicken genome, clusters of repeated DNA sequences occur which have alternate arrangements of the component sequence elements. Many of these clustered, repeated sequences are extensively methylated. We have established that both their arrangement and their methylation are invariant regardless of the source of chicken DNA. Comparisons included DNA from sperm, from a series of embryonic stages, from tissues of single adult individuals, and from thirty individual chickens of two strains. These same sequences are found in the DNA of some avian species related to chickens, and there they show the same clustered, methylated form. In related species, some of the arrangements found in chicken DNA are different or missing.     

Two-step recruitment of RNA-directed DNA methylation to tandem repeats

Tandem repeat sequences are frequently associated with gene silencing phenomena. The Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence is likely important. Endogenous FWA can adopt either of two stable epigenetic states: methylated and silenced or unmethylated and active. Surprisingly, we found small interfering RNAs (siRNAs) associated with FWA in both states. Despite this, only the methylated form of endogenous FWA could recruit further RNA-directed DNA methylation or cause efficient de novo methylation of transgenic FWA. This suggests that RNA-directed DNA methylation occurs in two steps: first, the initial recruitment of the siRNA-producing machinery, and second, siRNA-directed DNA methylation either in cis or in trans. The efficiency of this second step varies depending on the nature of the siRNA-producing locus, and at some loci, it may require pre-existing chromatin modifications such as DNA methylation itself. Enhancement of RNA-directed DNA methylation by pre-existing DNA methylation could create a self-reinforcing system to enhance the stability of silencing. Tandem repeats throughout the Arabidopsis genome produce siRNAs, suggesting that repeat acquisition may be a general mechanism for the evolution of gene silencing.     

Profiles of DNA methylation in normal and cancer cells

In eukaryotes, the epigenetic mark DNA methylation is found exclusively at cytosine residues in the CpG islands of genes, transposons and intergenic DNA. Among functional roles, DNA methylation is essential for mammalian embryonic development, and is classically thought to function by stably silencing promoter activity. However, until recently, understanding of the distribution of cytosine methylation in the whole genome - and hence, identification of its targets - was very limited. High-throughput methodologies, including methylated DNA immunoprecipitation, have recently revealed genome-wide mapping of DNA methylation, and provided new and unexpected data. Clearly DNA methylation is selectively associated with some key promoters- and is not a prerequisite for promoter inactivation, since strong CpG island promoters are mostly unmethylated, even when inactive. Most germline-specific genes are methylated and permanently silenced in somatic cells, suggesting a role of this mark in maintaining somatic cellular identity. These large scale studies will also help understanding the deregulation of DNA methylation associated with cancer, among which unmethylation of germinal cells genes, and recent observtion of large hypomethylated regions in tumoral specimens. The next challenge will be to understand if these methylation changes occur randomly, or more likely are specified by oncogenes or linked to environmental pressure.     

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

Background  

Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.