It appears that the following data and interpretations presented in this review have stood the test of time. Research, of course, is a never-ending enterprise, and conclusions have always to be considered subject to change as new data and concepts are being adduced. Here is a synopsis of concepts on the biological significance of m5C in the genome the author feels reasonably certain about at the time of this writing (December 2004).
The virus particle (virion)-encapsidated genomes of most mammalian DNA viruses are not methylated. Likewise, cellular DNA haphazardly integrated into an adenoviral genome, which becomes virion enclosed, does not become methylated, irrespective of its methylation status in the genome it has been derived from. In contrast, the DNA of frog virus 3, an iridovirus, is extensively, probably completely, methylated.
The concept of sequence-specific promoter methylations being causally related to long-term gene silencing, which has been first deduced from work on adenovirus promoters, has proved to be generally applicable in most eukaryotic genomes. Frog virus 3 promoters are an interesting exception to this apparent rule. Concomitantly with promoter methylation, histone modifications and perhaps modifications of additional proteins involved in chromatin structure play a decisive role in the regulation of promoter activity. At this time, it seems undecided whether DNA or protein modifications initially orchestrate these regulatory processes. It is likely that a refined interplay between both biochemical mechanisms comes close to the correct answer.
Foreign DNA, which has become integrated into an established mammalian genome, becomes de novo methylated in distinct patterns. The sites of initiation of de novo methylation at least in integrated Ad12 genomes are located paracentrally in the transgenomes and not close to the junctions with cellular DNA. In integrated Ad12 genomes, this localization of methylation initiation sites might be influenced by the transcriptional activity of the terminally located E1 and E4 regions of the Ad12 genome in the transformed cell lines or in Ad12-induced tumors, which are selected for the genetic activity of these viral genome segments. In any event, subsequent to initiation, de novo methylation extends continuously across the transgenomes in a spreading reaction. Initiation seems regional and does not emanate from a specific 5´-CG-3´ dinucleotide.
It is likely that hypermethylated or rather completely methylated transgenomes are more stably integrated than less completely methylated foreign DNA molecules. At the immediate sites of foreign DNA insertion, the patterns of cellular DNA methylation can be altered extensively.
Alterations of cellular DNA methylation patterns are, however, not restricted to the cellular junction sites [137, 138] but involve remote areas of the recipient genomes, even on different chromosomes. This trans effect is most striking in retrotransposon sequences, like the endogenous IAP DNA sequences in hamster cells, but can affect genuine cellular sequences as well. These remote perturbations of methylation patterns are not only observed after the integration of Ad12 DNA, which is partly transcriptionally active, but also after insertion of transcriptionally inactive bacteriophage lambda genomes. Possibly, ancient retrotransposons might be more responsive to local alterations of chromatin structure due to foreign DNA insertions into the recipient genome. There is evidence that in addition to alterations of methylation patterns in trans, the insertion of foreign DNA could also alter transcriptional patterns in the recipient genomes.
Many of the notions summarized here hold true not only for mammalian organisms but also for other eukaryotic genomes, particularly for those of plants.
In mammalian genomes, distinct patterns in the distribution of m5C residues exist which, at least in humans, can be interindividually preserved in several (many?) genome segments. These patterns are specific for each genome segment and can be different from cell type to cell type. These observations constitute a major challenge to the so-called Human Epigenome Project.
The biological importance of these patterns, which have obviously been conserved over long periods of time, has not been clarified. Long-term gene silencing and chromatin structure as well as the defense against foreign retrotransposons may be factors of significance in explaining the nature of these patterns of genome methylation.
Many members of my laboratories in Koeln (1972 to 2002) and in Erlangen (2002 to the present) have essentially contributed to the data summarized in this article. Their work has been acknowledged in the references cited herein. I am indebted to Petra Böhm, Koeln, for expert editorial work.
During different times, our research on DNA methylation has been made possible by grants and/or support from the following organizations: Deutsche Forschungsgemeinschaft, Bonn - SFBs 74 and 274; Center for Molecular Medicine in Cologne (CMMC, TV13); Federal Ministry for Research and Technology (BMFT), Bonn; Amaxa GmbH, Köln; Bayerisches Staatsministerium für Landschaftsentwicklung und Umwelt-gestaltung, München; Alexander von Humboldt-Stiftung, Bonn; Thyssen Stiftung, Köln; Sander Stiftung, München; Fonds der Chemischen Industrie, Frankfurt; European Union, Bruxelles; Universität zu Köln; Institut für Klinische und Molekulare Virologie, Erlangen.
It is a great pleasure to contribute this chapter to a volume of Biochemistry (Moscow) published in honor of Professor Boris F. Vanyushin on the occasion of a special birthday. Over a period of more than 20 years, I have valued our personal and scientific friendship, which started at a time when scientists of our countries had to devise special tools to be able to meet.
Accept my cordial greetings, Boris Fedorovich!
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