Eukaryotic DNA Methylation Research Articles

Erratum.

Eukaryotic DNA methylation as an evolutionary device. Vincent Colot and Jean-Luc Rossignol. (Article was originally published in BioEssays, Volume 21, No. 5, 1999.) In Figure 1, the term "Fungi" was positioned incorrectly. The correct version of Figure 1 appears here.

Eukaryotic DNA methylation as an evolutionary device.

DNA methylation is catalyzed by a family of conserved DNA methyltransferases and is widespread among protists, plants, fungi and animals. It is however absent in some species and its genomic distribution varies among organisms. Sequence comparisons suggest that known and putative eukaryotic DNA methyltransferases fall into at least five structurally distinct subfamilies. Furthermore, it is now clear that DNA methylation can be involved in several functions, some of which may coexist within the same organism. It can inhibit transcription initiation, arrest transcript elongation, act as an imprinting signal, and suppress homologous recombination. On the basis of these observations, we argue that DNA methylation has been conserved during evolution because it provides unique possibilities for setting up functions of various types.

Eukaryotic DNA methylation as an evolutionary device

Eukaryotic DNA methylation as an evolutionary device

Light-Regulated Transcription of Genes Encoding Peridinin Chlorophyll a Proteins and the Major Intrinsic Light-Harvesting Complex Proteins in the DinoflagellateAmphidinium carterae Hulburt (Dinophycae)1

In the dinoflagellate Amphidinium carterae, photoadaptation involves changes in the transcription of genes encoding both of the major classes of light-harvesting proteins, the peridinin chlorophyll a proteins (PCPs) and the major a/c-containing intrinsic light-harvesting proteins (LHCs). PCP and LHC transcript levels were increased up to 86- and 6-fold higher, respectively, under low-light conditions relative to cells grown at high illumination. These increases in transcript abundance were accompanied by decreases in the extent of methylation of CpG and CpNpG motifs within or near PCP- and LHC-coding regions. Cytosine methylation levels in A. carterae are therefore nonstatic and may vary with environmental conditions in a manner suggestive of involvement in the regulation of gene expression. However, chemically induced undermethylation was insufficient in activating transcription, because treatment with two methylation inhibitors had no effect on PCP mRNA or protein levels. Regulation of gene activity through changes in DNA methylation has traditionally been assumed to be restricted to higher eukaryotes (deuterostomes and green plants); however, the atypically large genomes of dinoflagellates may have generated the requirement for systems of this type in a relatively "primitive" organism. Dinoflagellates may therefore provide a unique perspective on the evolution of eukaryotic DNA-methylation systems.

DNA Methylation in Eukaryotes

In prokaryotes, DNA methylation protects the bacteriaÕs DNA against degradation by restriction enzymes and corrects error in DNA replication by means of the mismatch repair system. In 1964, the first DNA methyltransferase was identified in E. coli. The avaiability of sequence-specific restriction enzymes and methyltransferases led to a major breakthrough in the field of molecular biology. In eukoryotes, DNA methylation appears to have a different role. It has been implicated in the control of several cellular processes, including differentiation, gene regulation and embryonic development. A series of discoveries over the past few years has generated new interest in DNA methylation. It has been found that in mice in particular DNA methyltransferase is essential for normal embryonic development. Cytosine methylation may also contribute to C-T transition mutations, accounting for about one-third of all somatic and germline mutations in humans.

DNA methylation in eukaryotes

Recent advances have expanded our understanding of the processes underlying the establishment, maintenance, and elaboration of DNA methylation patterns in eukaryotes. The functional significance of DNA methylation is sought in a comparison of results on a variety of epigenetic phenomena in different eukaryotes. The recent development of DNA methylation mutants in mice, Neurospora, and Arabadopsis will allow traditional genetic dissection to be applied to long-standing problems regarding the function and regulation of eukaryotic DNA methylation. Although methylation appears to be important for maintenance of different epigenetic states, the mechanism that establishes these states is likely to involve additional processes.

Uptake of foreign DNA by mammalian cells via the gastrointestinal tract in mice: methylation of foreign DNA--a cellular defense mechanism.

Many reviews have been published on eukaryotic DNA methylation, but in spite of intensive work for more than two decades, many of the biological implications of this genetic signal, by some termed an “epigenetic signal”, still remain to be elucidated. The nucleotide 5-methyldeoxycytidine (5-mC) has been recognized as a modulator of DNA motif-protein interactions and hence has the potential to affect many reactions of the DNA molecule. For my own motivation to continue research in this fascinating yet complex field of investigations, the finding has been decisively stimulating that in distinct, randomly selected segments of the human genome patterns of DNA methylation are identical at the nucleotide level or in larger regions among different individuals of diverse ethnic backgrounds (Kochanek et al. 1990, 1993; Behn-Krappa et al. 1991). Therefore, it is highly unlikely that patterns of DNA methylation in established mammalian genomes are the result of a randomly acting DNA methyltransferase system. The available evidence indicates that patterns of DNA methylation are species-, cell type- and DNA segment-specific and vary with stage of development. We do not yet know how these patterns arise, whether they can fluctuate, or what their functional significance may be. Most researchers in the field agree that motif-specific methylation serves an important function in the long-term silencing of eukaryotic promoters (Doerfler 1983).

Abnormal Chromosome Behavior in Neurospora Mutants Defective in DNA Methylation

The function and regulation of DNA methylation in eukaryotes remain unclear. Genes affecting methylation were identified in the fungus Neurospora crassa. A mutation in one gene, dim-2, resulted in the loss of all detectable DNA methylation. Abnormal segregation of the methylation defects in crosses led to the discovery that the methylation mutants frequently generate strains with extra chromosomes or chromosomal parts. Starvation for S-adenosylmethionine, the presumed methyl group donor for DNA methylation, also produced aneuploidy. These results suggest that DNA methylation plays a role in the normal control of chromosome behavior.

Developmental differences in methylation of human Alu repeats.

Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.

Developmental differences in methylation of human Alu repeats.

Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.

Topological properties of bovine papillomavirus type 1 (BPV-1) DNA in episomal nucleoprotein complexes: a model system for chromatin organization in higher eukaryotes.

Sedimentation analysis of isolated episomal bovine papillomavirus type 1 (BPV-1) nucleoprotein complexes in sucrose gradients and subsequent separation of the purified DNA in chloroquine gels revealed different classes of molecules, varying in their degree of superhelicity. Since torsionally stressed DNA favors the adoption of secondary structures, we employed the single-strand-specific S1 nuclease to detect such structural alterations in both naked DNA and native chromatin. Direct examination of nuclease digestion products in chloroquine gels showed that neither the naked DNA nor the BPV-1 nucleoprotein complexes in isolated nuclei were cleaved randomly by the enzyme. Instead, there was a strict dependence on nuclease susceptibility and the degree of supercoiling, strongly suggesting that the structural features detected by S1 nuclease are due to the occurrence of torsionally stressed viral chromatin. Mapping analysis using the indirect end-labeling method demonstrated an S1-nuclease cleavage site adjacent to 20 homopurine residues known to be hypersensitive to S1 attack. Furthermore, direct methylation experiments with viral chromatin in isolated nuclei indicated that only circular, covalently closed nucleoprotein complexes served as substrate, whereas linearized BPV-1 chromatin was not susceptible to exogenously added Hhal methylase. This observation raises the possibility that the modulation of topology in nucleosomally organized DNA might also play a role in eukaryotic DNA methylation.

DNA methylation and chromatin structure: a view from below

An understanding of the function and control of DNA methylation in eukaryotes has been elusive. Studies of Neurospora crassa have led to a model that accounts for the chromosomal distribution of methylation and suggests a basic function for DNA methylation in eukaryotes.

DNA methylation in eukaryotes: kinetics of demethylation and de novo methylation during the life cycle.

We present a model for the kinetics of methylation and demethylation of eukaryotic DNA; the model incorporates values for de novo methylation and the error rate of maintenance methylation. From the equations, an equilibrium is reached such that the proportion of sites which are newly methylated equals the proportion of sites which become demethylated in a cell generation. This equilibrium is empirically determined as the level of maintenance methylation. We then chose reasonable values for the parameters using maize and mice as model species. In general, if the genome is either hypermethylated or hypomethylated it will approach the equilibrium level of maintenance methylation asymptotically over time; events occurring just once per life cycle to suppress methylation can maintain a relatively hypomethylated state. Although the equations developed are used here as framework for evaluating events in the whole genome, they can also be used to evaluate the rates of methylation and demethylation in specific sites over time.

Differential regulation of DNS methylation in rat testis and its regulation by gonadotropic hormones

Eukaryotic DNA methylation occurs exclusively at the 5'-position of cytosine and has been implicated in the regulation of gene expression. Using high-performance liquid chromatography, the methylation of testis DNA during its development, in different cell populations and during regulation by gonadotropic hormones, were studied. The 5-mC content of testis DNA increased significantly from days 30 to days 150, while in 2-yr-old testis 5-mC content decreased significantly. Among various populations of testicular cells, pachytene spermatocyte DNA contained a significantly high amount of 5-mC when compared to spermatogonia, spermatids and mature sperm DNA However, the 5-mC content of elongated spermadids was significantly less when compared to the above four fractions. Administration of follicle stimulating hormone to immature rats caused hypomethylation of seminiferous tubular DNA while luteinizing hormone caused similar effects in Leydig cells. These results indicate that in testis, DNA methylation is differentially regulated during development and is controlled by gonadotropic hormones.

Eukaryotic DNA methylation and demethylation--sequence and strand specificity.

Eukaryotic DNA methylation and demethylation--sequence and strand specificity.

Sister chromatid exchanges induced by DNA demethylating agents persist through several cell cycles in mammalian cells

Eukaryotic DNA methylation has been extensively studied in recent years. The ability of many carcinogens to interfere with DNA methylation has not yet been directly related to their tumorigenic activity. Recent data obtained using L-ethionine and 5-azacytidine--both demethylating agents--showed a small but significant increase in the sister chromatid exchange (SCE) rate induced in mammalian cells (human lymphocytes and CHO cells). In this paper we show that the SCE increase induced by both these agents in Chinese hamster ovary (CHO) cells persists for as long as 10 cell cycles. On the other hand mitomycin-C and u.v. light-induced SCEs show a rapid decrease to the control value, as reported for all known SCE inducers. We suggest that DNA demethylation and SCEs are connected through a perturbation of the cell machinery at the level of the replication fork, producing an increase of the error-prone ligation. Since the methylation level is maintained (inherited), the SCE increase produced by these recombinational events will not be corrected through several cell cycles.

Eukaryotic DNA methylation

Eukaryotic genomes contain 5-methylcytosine (5mC) as a rare base.5mC arises by postsynthetic modification of cytosine and occurs, at least in animals, predominantly in the dinucleotide CpG. The base is not distributed randomly in these genomes but conforms to a pattern. This pattern varies between taxa but appears to be inherited in a semi-conservative fashion. At the level of the genome, gross changes in the level of DNA methylation have been noted. This has encouraged speculation that the modification may play a role in cellular differentiation. Tissue-specific patterns of DNA methylation, predicted by various models of differentiation, have been found for most vertebrate genes so far examined. A correlation has emerged between the undermethylation of these regions and their transcription, but this is not always the case. While data for eukaryotic viral sequences are less equivocal, studies of this kind cannot in isolation distinguish between undermethylation being a cause or a consequence of gene activity. If it were a cause, it is probable that the demethylation of specific CpG sites would be a necessary yet not a sufficient condition for transcription to occur. The introduction of artificially methylated DNA sequences into individual eukaryotic cells by microinjection or transformation may provide the means to elucidate these questions in the future. In the meantime, the study of eukaryotic DNA methylation promises to contribute much to our understanding of the regulation of gene expression in these organisms.

Demethylation of CpG sites in DNA of early rabbit trophoblast.

The function of DNA methylation in eukaryotes is unknown. The introduction of methyl groups into the major groove of B-form DNA has been shown to affect DNA–protein interactions in prokaryotes1, and it is probable that they perform analogous functions in eukaryotic DNA. Thus, there is considerable interest in the possibility that DNA methylation may be involved in regulating DNA transcription and in establishing stable patterns of genetic expression in higher organisms1,2. Restriction enzyme studies have shown that methylation of eukaryotic DNA is not random; methylation patterns are tissue specific when individual genetic loci are examined3–5. On the other hand, it is also apparent that these tissue-specific patterns tend to be rather subtle and to occur within overall levels of methylation that are relatively constant in all tissues of a given organism4. For example, no significant change in total DNA methylation has been detected during embryogenesis in either the sea urchin6,7 or the mouse8, even though rapid cellular differentiation is occurring. We present evidence here, however, for extensive changes in methylation accompanying early differentiation in the rabbit embryo. Whereas the DNA of the early cleaving embryo is highly methylated, as is that of the later embryoblast, the terminally differentiated trophoblast9 is demethylated by about 35%.

Detection of methylated sequences in eukaryotic DNA with the restriction endonucleases SmaI and XmaI

The restriction endonuclease XmaI cleaves eukaryotic DNA whether or not its recognition sequence (C ↓ -C-C-G-G-G) contains 5-methylcytosine in the CpG dinucleotide. This is in contrast to its isoschizomer, endo R SmaI, which does not cleave DNA if the CpG of its recognition sequence (C-C-C ↓ G-G-G) is methylated. Thus, this isoschizomer pair will be useful in the study of eukaryotic DNA methylation.

Use of restriction enzymes to study eukaryotic DNA methylation: II. The symmetry of methylated sites supports semi-conservative copying of the methylation pattern

Two experiments with Xenopus laevis ribosomal DNA have demonstrated that in the base-paired sequence ▪ either both cytosines are methylated or neither is methylated. Half-methylated sites are not found. The first experiment involved reassociation of a labelled methylated ribosomal DNA restriction fragment (4.65-RI) with an excess of the unmethylated fragment in order to expose any half-methylated sites to CpG-enzyme digestion. Subsequent treatment with HpaII and HhaI showed that methylated/unmethylated rDNA hybrids are no more sensitive to digestion than the native rDNA. The result indicates that fewer than 2% of methylated sites are half-methylated. In the second experiment erythrocyte rDNA was denatured and allowed to self-reassociate. This procedure rendered the 4.65 kilobase EcoRI fragment immune to digestion with HpaII, AvaI and HhaI at all sites except the HhaI hotspot. Since the native fragment was partially digested at many sites by HpaII, AvaI and HhaI prior to annealing, its subsequent immunity indicates that most paired CpGs are symmetrically methylated. The result also verifies the presence of a specific undermethylated recognition sequence for HhaI. Finally it has been shown for X. laevis cultured cells that following DNA replication new methyl groups are added to the progeny strand. The parental strand was not detectably labelled with [ methyl- 3H]methionine. Taken together these results indicate that any pattern of methylated and unmethylated paired CpGs in the genome of a cell will be inherited by descendants of that cell.