Functions of Eukaryotic DNA Methylation
By Jean-Luc Rossignol
Institut Jacques Monod, UMR 7592 (CNRS/Univ. Paris 6/Univ. Paris 7)
Paris, France
Introduction
Cytosine methylation is a chemical modification of DNA which can be faithfully inherited. In contrast to base substitution mutations, this chemical modification does not change the pairing properties of the modified base. C-methylation is observed in the three domains of life: Archae, Bacteria and Eucaryotes. It is catalyzed by C-DNA methyltransferases (MTases) which all share a catalytical domain with nine well-conserved motifs arranged in the same order along their primary structure. This indicates that C-methylation is an ancestral mechanism that predated the diversification of the three domains of life.
However, methylation is not observed in all eukaryotes. Whereas it has been found in all plants and vertebrates studied, it has not been detected in several unrelated animal species. In fungi, it is present in some species (Ascobolus immersus, Neurospora crassa) but has not been detected in others (Saccharomyces cerevisiae, Schizosaccaromyces pombe). The failure to find any gene related to MTases in the completely sequenced genomes of C. elegans and S. cerevisiae strongly suggests that methylation is actually absent in these organisms.
How can we solve this apparent paradox? On the one hand, methylation is an ancestral process which has been conserved in many animal, plant and fungal species despite its mutational load (deamination of methylated Cs favours C to T transitions, hypermethylation of gene suppressor promoters favors progression to cancer). On the other hand, methylation is absent in various fungal and animal species. This clearly means that methylation does not deserve any specific essential function which would be common to all eukaryotes. We have proposed, with Vincent Colot (1) that methylation has been maintained during evolution, because, owing to its property to provide an inheritable marking to DNA, it acts as an evolutionary device which has been used for setting up a variety of functions.
In support of this hypothesis, methylation displays distinct patterns of distribution among eukaryotic genomes, several types of MTases have been identified which can be classified in distinct subfamilies and methylation serves a variety of functions which can either coexist within the same type of organism or differ from one type to another.
1) Diversity of methylation patterns
The distribution of methylation varies widely among eukaryotic genomes (1, for a review). In vertebrates, methylation is genome wide, involving all genomic regions, with the exception of the one-two kb long DNA segments named “CpG islands.” CpG islands are mainly located upstream from the genes and are found in about two third of them. They display a high density of CpG doublets, as compared to the rest of the genome. These CpGs usually stay unmethylated, with a few exceptions, like in the inactivated X of mammalian females. In contrast, in plants and fungi, methylation is fractional, being mainly located in genomic regions corresponding to DNA repeats created by transposons and retrotransposons. However, this is not the only pattern of fractional methylation. In the Urochordate Cyona intestinalis, methylation was found within the transcribed part of single genes, but was absent from DNA repeats (2).
Another factor contributes to the variety of methylation patterns: it corresponds to nature and the density of methylation substrates (1, for a review). In adult mammalian tissues, methylation involves almost only Cs belonging to symmetrical CpG doublets. Furthermore, with the exception of CpG islands, the density of CpGs is about three times lower than expected from the density of C and G. In contrast, in fungi, CpGs are as frequent as expected, and methylation also involves Cs which do not belong to CpGs. Therefore, in methylated genomic regions, the density of methylation is much higher in fungi than in vertebrates.
2) Multiplicity of eukaryotic methyltransferases
By comparing the nine most conserved motifs of the catalytical part of MTases, it is possible to set up a phylogenetic tree (Figure). There is as much diversity between eukaryotic Mtases as between eukaryotic and prokaryotic DNA MTases. Eukaryotic MTases can be tentatively classified in five subfamilies. In two of these, the three kingdoms: animals, plants and fungi are represented. These observations suggest that the diversification of MTases predated the appearance of these eukaryotic kingdoms.
Figure : Phylogenetic
tree of eukaryotic C-DNA methyltransferases
3) Various functions of methylation
The present experimental evidence indicates that methylation can serve at least four distinct functions: either preventing or favouring gene expression, marking imprinted genes and repressing recombination.
3.1. Repressing gene expression
In mammalian cells, a strong correlation is found between the repression of the transcription of genes located downstream CpG islands and the methylation of these CpG islands. In vitro-methylation prevents the expression of genes after transfection. There is now good evidence that methylation of promoters containing a high amount of CpGs has also a causal role in preventing transcription initiation in vivo (see 1 for a review, and 4). Methylation would allow the establishment and maintenance of inactive chromatin states owing to the recruitment of histone-deacetylases either through methyl-binding proteins (1) or through MTases which are targeted to the replication fork during the late S phase (see 5 for a review).
In fungi also, methylation inhibits transcription, but this is obtained via an effect upon transcript elongation, which is blocked when it reaches methylated portions. In contrast, little or no effect is observed on transcription initiation. In contrast, in mammals, heavy methylation of the transcribed sequence does not appear to affect the transcript elongation.
Therefore, methylation represses transcription in two completely different ways in vertebrates and in fungi, by blocking its initiation in the first class of organisms and by preventing transcript elongation in the second one.
3.2. Marking imprinted genes
Another function of methylation is to provide an heritable signal which has no direct effect upon transcription. This is illustrated by the situation commonly observed in the case of parental imprinting in mammals. Imprinted genes display a monoallelic expression restricted to the paternal allele (for some imprinted genes) or the maternal allele (for some others). The imprint occurs in the germline and has been shown in several cases to be associated with the uniparental methylation of a small DNA region lying close to the imprinted gene, called the “imprinting box.” Several observations indicate that methylation acts as a signal that can be dissociated from the effect upon gene expression. Indeed, the monallelic expression is set up only in the post-implantation embryo, after numerous cell divisions, which implies that the signal was inherited without being executed. Furthermore, in mutants cells which do not express the maintenance MTase dnmt1, the methylation of the imprinting box is lost and monoallelic expression is never set up. Introducing a functional dnmt1 gene by transfection does not enable the restoration of the monoallelic expression, indicating that it is indeed the loss of a transmissible signal which is responsible for the lack of monoallelic expression rather than a direct consequence of the absence of methylation (see 1 for a review).
3.3. Allowing gene expression
The allele, which will stay unexpressed when monoallelic expression is set up, can be the allele which underwent methylation of its imprinting box. This can be simply explained if, after implantation, methylation expanded to the promoter region of the imprinted gene, hence preventing its expression. However, the other situation where the expressed gene is the one that underwent methylation of its imprinting box is also observed. In this situation, both alleles stay unexpressed during early development, and the loss of the imprinting signal in MTase deficient cells leads to a failure of expression of the imprinted gene, resulting in both alleles being silenced. This means that, in this case, the methylation of the imprinting signal is required to trigger gene expression.
An example of this situation is seen with Igf2, in mouse. H19 and Igf2 are two linked imprinted genes. The expression of H19 is restricted to the maternal chromosome, whereas that of Igf2 is restricted to the paternal one. The imprinting box is methylated on the maternal chromosome, and the loss of the imprinting methylation signal results in the absence of expression of Igf2. In other words, the expression of Igf2 requires the methylation of the imprinting box. The teams of G. Felsenfeld and S. Tilgham (reviewed in 6) have shown that the imprinting box corresponds to an insulator element. The insulation is mediated by the protein CTCF which binds this region. On the unmethylated maternal chromosome, an enhancer element located close to H19 activates this gene. The presence of the insulator, distal to H19, prevents the enhancer from activating Igf2, located farther away. In contrast, on the paternal chromosome, methylation of the imprinted box prevents the binding of CTCF, and this region can no longer act as an insulator. Consequently, the enhancer can activate the distal Igf2 gene. On another hand, methylation of the imprinting box, which extends to the promoter of H19, prevents the expression of this gene.
3.4. Repressing homologous recombination
It was already shown that methylation of the immuglobulin genes prevented their rearrangement via the VDJ specialized recombination machinery, probably by inducing a close chromatin state which prevents the access of the recombinase (7). Methylation might also prevent homologous recombination. Indeed, in plants and fungi, methylation mainly affects DNA repeats, which often lie at distinct positions on the same or on distinct chromosomes. Events of homologous recombination between these repeats threaten genome integrity by producing various types of chromosome rearrangements. In this regard, methylation might contribute to genome stability by preventing homologous recombination.
In order to test this hypothesis in our laboratory, Laurent Maloisel made a construct in the fungus Ascobolus in which a meiotic recombination hot spot flanked by two genetic markers was integrated within a chromosome. The frequency of crossing-over was measured in strains in which the hot spot was either unmethylated, methylated in both parental DNA molecules or methylated in only one DNA molecule. Recombination dropped by about 300 times in the strains where both parental molecules had been methylated and by near 50 times in strains harboring one methylated and one unmethylated parent molecules (8). Studies in yeast have shown that meiotic recombination is induced by a double-strand break involving either parental molecule with equal probability. If methylation were to only prevent the initial break, we would expect methylation to suppress only half of the recombination events. The 50 times decrease therefore indicates that methylation acts at steps of the recombination process other than just the initial cut.
Conclusion
Besides the fact that it is not universal among eukaryotic organisms, methylation displays a large diversity of genomic pattern distributions, is catalyzed by a variety of MTases, and performs distinct functions. These functions can coexist within the same organism (either repressing or allowing transcription and being a signal for parental imprinting in mammals; repressing both transcription and recombination in fungi). They also can differ from one type of organism to another one (for example, preventing transcript initiation in vertebrates and arresting transcript elongation in fungi). This variety in methylation conditions, distribution and function is expected in our hypothesis of methylation being used during evolution as a molecular device to set up new functions. It is likely that in the near future, new functions of eukaryotic methylation will be deciphered.
References