On the Biological Significance of DNA Methylation
Institut für Genetik, Universität zu Köln, Zülpicherstr. 47, 50674 Köln, Germany; E-mail: [email protected]
Institut für Klinische und Molekulare Virologie, Universität Erlangen, Schloßgarten 4, D-91054 Erlangen, Germany; E-mail: [email protected]
This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past thirty years. The text does not attempt to give a complete and meticulous account of the many relevant and excellent reports published by many other laboratories, so it is not a review of the field in a conventional sense. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proven their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (i) the de novo methylation of integrated foreign genomes; (ii) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (iii) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (iv) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (v) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; what role has food-ingested DNA played in the elaboration of this mechanism?
KEY WORDS: DNA methylation, adenoviruses, cancer
The results of research on the biochemistry and biology of DNA methylation have grown into a sizable body of scientific information. A single article like this one can, of course, not even attempt to present an adequate overview of this rapidly developing field. This chapter has therefore been restricted to a synopsis of selected work performed in the author's laboratory between 1975 and 2004.
For a long time, many colleagues in molecular biology resisted recognizing the fact that the fifth nucleotide in DNA, 5-methyl-deoxycytidine (m5C), exerts decisive functions in chromatin structure and in genetic control mechanisms. With m5C, however, the arguments have finally become irrevocably strong enough not to be ignored. Nevertheless, text books still preach the existence of four, instead of five, nucleotides in DNA. Of course, it is good and essential scientific practice to cast most critical scrutiny on new claims and demand ample and definitive experimental proof. A large number of researchers have now provided this proof, and many of the findings will be summarized in this volume. My own group started contributing to the honing of problems related to DNA methylation in the mid-1970s, and this article presents a detailed summary of our results which have been adduced since then and stood the test of time. For further information, the reader can consult the references cited herein and previous reviews, which have been published as our work proceeded [1-7].
The discovery of m5C  in eukaryotic, particularly in mammalian, DNA has provoked a challenging search for its functional significance. This search is by no means completed, and active investigations on numerous unsolved questions are still continuing. The modification of cytidine (C) to m5C, apparently the only one among the nucleotides in mammalian DNA, is introduced postreplicationally by several DNA methyltransferases (DNMT) which are chosen depending on the functional context of their enzymatic activity: DNA can be methylated de novo, still a most enigmatic series of events, or a given pattern of DNA methylation in the genome can be maintained upon replication. In this latter mode of maintenance methylation, the parental DNA strand with the m5C residue still in place can serve as the template to direct the DNMT to modify the newly synthesized DNA complement. Although several DNMT have been well characterized, it is not clear whether any of them by itself suffices to facilitate either of the two modes of DNA methylation. In addition to the enzymatic activity proper, the function of these enzymes seems to depend critically on the conformation of the local chromatin segment in which the DNA is to be methylated. Since our understanding of chromatin structure is incomplete, we cannot expect to obtain a comprehensive description of the enzymatic activities of the DNMT. It appears more realistic to propose a complex interplay between DNA-chromatin structure and specific choices of enzymatic functions in which additional regulatory proteins have to participate.
In experimental terms, DNA methylation activities cannot be realistically assessed by relying on the measurement of enzymatic function using a naked DNA template, since the actually operational template for DNMT is a DNA-chromatin complex with site-specific, stochastically malleable functions which are targeted to individual loci in the genome. It will be some time before these processes can be elucidated or even mimicked by current technology. How can we approach a functional analysis of DNA methylation in eukaryotic, particularly in mammalian, systems? One important parameter in understanding this functional DNA modification is to realize that m5C residues are not introduced randomly by a fortuitously acting enzymatic mechanism. In contrast, highly specific patterns in the distribution of m5C residues exist all over the genome. These patterns appear to be different in each cell type and in each region of the genome. It will require a major effort to determine these patterns of DNA methylation in all parts of the mammalian, specifically in the human, genome.
In recognizing the very significant accomplishment of determining the nucleotide sequence of the human genome, I submit that the task has not been completed without the inclusion of the fifth nucleotide. Of course, it is technically impossible to differentiate between a C- and a m5C-residue by the conventional sequencing reaction. The application of the bisulfite protocol of the genomic sequencing reaction [9, 10] is a demanding project, particularly when it has to be extended to many kilobases of DNA sequence. Nevertheless, this method is, at least for the time being, the only reliable procedure to ascertain levels and patterns of DNA methylation. By applying the bisulfite reaction, one can detect all m5C residues in a sequence. The human epigenome project has just been initiated on an exploratory basis and will have to cope with the fact that patterns of DNA methylation can be different from cell type to cell type and, of course, in each segment of the genome . In my laboratory, we have investigated methylation patterns in several areas of the human genome to obtain a first impression of the types of patterns. The structure of the genome inside its chromatin casing and its regulatory functions appear to depend on these patterns of DNA methylation. The function of the genome will not be understood before the completion of the analysis of these patterns. Hence, the study of more complex biomedical problems will undoubtedly escape a thoroughly informed experimental approach before this analysis has been finished. Presently available data imply that in 90 human genes in the major histocompatibility complex (MHC) of multiple tissues and individuals, the majority of regions were hypo- or hypermethylated. The patterns were tissue-specific, interindividually variable, and correlated with gene expression .
The following Gedankenmodell may aid the conceptual visualization of a more general function of patterns of DNA methylation across the entire genome. The model is based upon the notion that m5C residues are modulators of DNA-protein interactions, as proposed earlier , and these modulators could facilitate and enhance or abrogate such interactions. The direction, in which these modulations work, depends on the type of protein and DNA sequences in functionally crucial interactions.
Imagine a bare wall represented here by the plain nucleotide sequence of A, C, G, and T residues onto which elaborate decorations have to be attached. Chromatin proteins then are the decorations, which eventually contribute to the chromatin structures and could be specific for different segments of the genome. Now, we insert into the blank wall the m5C “pegs” to which proteins bind or are prohibited from binding. With this first and essential set of DNA-protein interactions, a central genome-associated scaffold will be generated which then will be able to inaugurate further protein and/or RNA assemblies until the final, yet enigmatic, chromatin structure has been established.
Local specificities in this structure will, of course, be determined by the site-specific pattern of DNA methylation, which thus assumes a functionally crucial role in this assembly process. There are several, but one particular, problem with this model: it is not apparent whether the generation of a given pattern of DNA methylation arises before or after chromatin formation. Possibly, both events are interdependent and develop concomitantly. Upon DNA replication, an established and inheritable pattern of DNA methylation is, of course, maintained by the array of m5C residues, which are still preserved after DNA replication in the parental strand and which can serve as a template for the insertion of methyl groups in the newly synthesized DNA complement. In this way, patterns of DNA methylation are propagated and inherited. The methylation patterns in turn promote the site-specific chromatin structures.
A further tantalizing aspect arises from the fact that DNA methylation patterns are erased early in embryonic development and are thereupon re-imposed by an unknown mechanism of de novo DNA methylation, which cannot avail itself of the template pattern on the complementary strand of DNA. Conversely, the fixation of de novo methylation patterns on integrated foreign DNA or in the course of embryonic development might be directed by local chromatin structures, which then would have to be “remembered” even in the absence of the fifth nucleotide. It is this crucial interdependence between methylation pattern and chromatin structure that we cannot yet satisfactorily explain. RNA could conceivably serve as a mediator for this functional gap in time and structure. This model is based on the fact that each individual segment of the genome is tightly associated with a given pattern of DNA methylation and, consequently, of chromatin structure. The same or a very similar site-specific pattern can also be conveyed to foreign DNA subsequent to its insertion into a specific segment of the mammalian genome.
In part, this model has been deduced from the observation that the site-specific re-integration of an unmethylated mouse gene, the B lymphocyte tyrosine kinase (BLK) gene, into the mouse genome by homologous recombination leads to the re-establishment of the original and authentic DNA methylation pattern in the integrate at its authentic site . In contrast, when the BLK gene randomly hits host DNA sequences and recombines there by a non-homologous mechanism, patterns of DNA methylation are completely different from the authentic pattern in the BLK gene. For a working hypothesis, we assume that each genome segment is characterized by a “methylation memory”. Its biochemical correlate is not known but must somehow be related to topical chromatin structure as well as local DNMT type, concentrations, and activities as well as auxiliary functions.
The most intensely studied function of DNA methylation in eukaryotic genomes is that of promoter activity and long-term gene silencing. Starting in the late 1970s, our laboratory has regularly contributed to the elaboration of this concept [1, 2]. In conjunction and - again in an interdependent mode - DNA (m5C) and chromatin (histone acetylation and methylation) modifications collaborate in the long-term silencing of promoters and thus assume an essential function in regulating the activity of specific genome segments. In recent years, these mechanisms have been recognized to be of importance also for the understanding of more complex biomedical problems, in particular those which are related to genetic imprinting, embryonic and fetal development, genetic disease, and tumor biology. Here, we have another fine example of how basic research on fundamental mechanisms in molecular genetics can eventually help understand practical problems in biomedical research. Without turning to the study of simpler experimental systems, e.g. viral models, in the elucidation of promoter silencing by DNA methylation and related histone modifications, it would have been impossible to approach more complex problems in mammalian organisms or in plants.
Source: Biochemistry (Mosc). 2005 May;70(5):505-24.