Microbial Pathogenesis 1992 ; 12 : 1-8

Mini-review DNA methylation : eukaryotic defense against the transcription of foreign genes? Walter Doerfler Institut fur Genetik, Universitat zu Koln, Weyertal 121, D-5000 Koln 41, Germany

Introduction The DNA of eukaryotes contains a fifth nucleotide, 5-methyldeoxycytidine (5-mC), whose concentration in DNA varies from a few per cent in mammalian DNA to more than 30% of all deoxycytidine residues in the DNA of plants . Frequently, 5-mC occurs in the dinucleotide combination 5'-CG-3' . It has been demonstrated for several segments of the human genome that 5-mC is distributed in highly specific patterns, which appear to be identical among different individuals even with different genetic backgrounds .' 2 Thus, it is very unlikely that the methylation of DNA is a random postreplicational event . On the contrary, patterns of methylation are cell-type-specific, perhaps species-specific, and are highly conserved . Patterns of DNA methylation are inherited through the mechanism of maintenance methylation . It is thought that 5-mC represents a potent genetic signal . The methyl group of 5-mC protrudes into the major groove of the DNA double helix and can be recognized by proteins . The prokaryotic restriction-modification system provides a well documented example and a framework for models on the functional significance of DNA methylation in eukaryotes . In prokaryotes, 5-mC in specific sequence combinations prohibits the action of several restriction endonucleases . In a few instances (e .g . Dpn I, NmuDl ), the presence of another modified base, N 6 -methyldeoxyadenosine (N 6 -mA), is required for the activity of these prokaryotic enzymes . A nucleotide sequence tagged by the presence of a methylated nucleotide can therefore function as a powerful modulator of DNA-protein interactions . The recognition of specific nucleotide sequences containing 5'-CG-3' dinucleotides by specific proteins can be compromised when 5-mC is present in the recognition sequence . On the other hand, cellular proteins have been identified that are targeted towards 5-mC residues ."' Since many of the important reactions in molecular biology are dependent on highly specific DNA-protein interactions, one can expect many biological processes to be influenced by the presence of 5-mC in specific sequence motifs . Species-, tissue-, and cell-type-specific patterns of DNA methylation may therefore assume the role of a functional regulator, and the analyses of patterns of DNA methylation may help to decipher these activity patterns . At present, we have only a fragmentary understanding of the biological significance of DNA methylation . It has been demonstrated that the sequence-specific methylation of promoter segments can lead to the long-term shut-down of gene activity . Since our laboratory has contributed to the documentation of this concept, I have been asked to write several reviews on this topic . 5-12 In the following section, I shall briefly summarize our own work on the adenovirus system . 0882-4010/92/010001 +08 $03 .00/0

n 1992 Academic Press Limited



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Integration of adenovirus DNA and de novo patterns of DNA methylation I have chosen the adenovirus system as a model for work on the biological significance of patterns of DNA methylation . 5-12 Virion DNA, i .e . the DNA molecule inside the adenovirus particle, is not detectably methylated . 13 Similarly, 5-mC residues cannot be traced in free viral DNA in productively 14 (C . Kammer and W . Doerfler, unpublished data) or in abortively infected cells ." Thus, in the intricate regulation of the viral transcription program during viral infection of permissive human or non-permissive hamster cells (for adenovirus type 12 = Ad12), DNA methylation is not likely to play a role . The DNA of adenoviruses can be integrated into the genomes of mammalian cells ." ," My laboratory was the first one to demonstrate the integration of Ad12 DNA into hamster DNA, 18• 1 9 and we have since pursued work on the mechanism of this apparently non-homologous insertion reaction .' 1,20-22 In particular, we have used clonal lines of adenovirus-transformed mammalian cells to investigate the structure of the sites of adenovirus DNA integration . Integrated adenovirus DNA in hamster cells is methylated in specific pattern s, 22-27 and this system has served as an example for the occurrence of de novo methylation of foreign DNA in mammalian cells . We have now demonstrated that the de novo methylation of foreign (adenovirus) DNA is initiated at sequences located internally in the integrated Ad12 genome and gradually spreads across most parts of the integrated Adl 2 genome . 27 Specific segments located at the termini of the Adl 2 DNA molecule do not in general become methylated . We have also shown that the cellular sequences adjacent to and abutting the integrated Ad12 genome are undermethylated in comparison to the same sequences prior to foreign DNA insertion .27 ,28 These findings suggest that the integration of foreign DNA can have profound effects on patterns of DNA methylation in the vicinity of the inserted foreign DNA . We do not know yet how far these changes can extend from the site of insertion into the cellular genome . It is conceivable that these alterations in patterns of DNA methylation, which have probably been evoked by inserting foreign DNA into a pre-existing genome, contribute .29 to the mechanism of cellular transformation by the oncogenic adenoviruses . 10,21 The enzymatic mechanism of de novo DNA methylation is still unknown . Several factors, such as the specific sequence of DNA, the site of foreign DNA integration, and the specificity of cellular DNA methyltransferases plus auxiliary factors, may play a role in eliciting very specific patterns of DNA methylation in the inserted foreign genome . 27 • 30 However, we do not understand yet how these very different factors can collaborate in the generation of de novo patterns of DNA methylation . De novo methylation differs mechanistically from maintenance methylation . This latter type of DNA methylation is responsible for passing on an established pattern of DNA methylation to future cell generations and explains why patterns of DNA methylation are strictly inherited . We do not know, however, whether in the generation of de novo patterns of DNA methylation the gradual spreading of methylation from one or a few 27,31,32 focal points can represent an important element .

Sequence-specific promoter methylation and gene inactivation In adenovirus-transformed cells, early viral genes are frequently transcribed, whereas the late genes are permanently silenced . In integrated Ad12 genomes in transformed hamster cells, we have observed inverse correlations between the extent of DNA methylation and the level of transcription ; the early viral genes are hypomethylated, the late genes strongly methylated . 23-25 Similar observations have been made in



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adenovirus type 2 (Ad2)-transformed cells ." In the ensuing years, we concentrated on studies with the late E2A promoter of Ad2 DNA and performed a number of model experiments in different systems which documented that the introduction of 5-mC into certain 5'-CG-3' sequences inactivated the late E2A promoter . We chose the positions +24, +6, and -215 in this promoter, relative to position +1 where transcription was initiated, because these sequences were methylated in cell lines in which this promoter was silenced, whereas they were unmethylated in cell lines that carried an active late E2A promoter ." Thus, we just imitated a state of promoter methylation in vitro that we had observed in living cells . Moreover, these sequences were part of 5'-CCGG-3' (Hpa I I recognition) sequences which could be methylated in vitro with a sequence-specific prokaryotic DNA methyltransferase . The methylation in vitro of the late E2A promoter at the designated sites silenced this promoter after microinjection into Xenopus /aevis oocytes, 33,34 after transfection into mammalian cells, 35 and after the genomic fixation of the methylated late E2A promoter in hamster cells . 36 Similarly, the promoter of the E1A gene in Ad12 DNA responded with inactivation, when the promoter was 5'-CCGG-3' or 5'-GCGC-3' methylated and transfected into mammalian cells ." The 5'-CCGG-3' methylated late E2A or major late promoter of Ad2 DNA also proved inactive or inhibited in a cell free transcription system from HeLa nuclear extracts, 38 and the late E2A promoter also after the genomic fixation in transgenic mice ." Promoter inhibition or inactivation by sequence-specific methylation is not unconditional . Without apparent demethylation to occur, the inhibitory effect can at least 35,39 partly be overcome by the 13S RNA gene prod uct, the 289 amino acid paradigm transactivator, 40 which is encoded by the E1A region of adenovirus DNA . A possibly similar transactivating function has been described in iridovirus, frog virus 3 (FV3)infected mammalian cells . 41 Promoter inhibition by DNA methylation can also be counteracted by the presence of a strong enhancer in the vicinity of the methylated late E2A promoter . 42 Promoter methylation is thus a pliable genetic regulatory signal that can be influenced by several other factors which are involved in the regulation of genetic activity . We have now set out to investigate by what mechanism promoter methylation can interfere with promoter functions . A number of possibilities can be envisaged as to how this genetic signal may modulate DNA-protein interactions . It is conceivable that strategically positioned methyl groups could directly prohibit the interaction of promoter motifs with transcription factors . Alternatively, proteins specialized in the binding of methylated motifs might interact with the methylated promoter sequence and in this way compromise the binding of other proteins essential for promoter function ." Detailed analytical work will be required to investigate this mechanism . The 5'-CCGG-3' methylated late E2A promoter has been examined by DNase I footprint analyses for differences in protein binding in comparison to the unmethylated promoter . None were found ." However, a 50 or 73 nucleotide, double-stranded oligodeoxyribonucleotide comprising promoter positions +37 to -13 or +69 to -4, respectively, formed a specific DNA-protein complex with nuclear extracts from HeLa cells . This complex contained several proteins, and the transcription factor AP2 was probably involved in the generation of this complex ." When these oligodeoxyribonucleotides were methylated in positions +24 and +6, i .e . in the downstream region of the promoter, complex formation was completely abrogated . The 50 nucleotide synthetic fragment competed for complex formation in the unmethylated, but not in the methylated form ." These results do not support the notion that a 5-mC recognizing protein binds to the methylated promoter positions in this oligodeoxyribonucleotide . On the other hand, definite conclusions cannot yet be drawn,



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since data gleaned from work with synthetic oligodeoxyribonucleotides need not represent the actual situation for the intact late E2A promoter .

Host defense mechanisms Excision of integrated foreign DNA? Discussions about possible host defense mechanisms in eukaryotic cells against the uptake and integration of foreign DNA can only be hypothetical at this stage . It is, however, an accepted point of view that in many prokaryotic organisms the restrictionmodification system can serve as a defense function against foreign, in particular viral, DNAs . In prokaryotes, extraneous DNA is cleaved sequence-specifically by the host's restriction endonucleases while the host DNA is protected against autodegradation by DNA-methyltransferases with corresponding sequence specificities . It is not known how frequently restriction endonuclease fragments of foreign DNA can be integrated into the genome of the prokaryotic host cells . An impressive variety of such enzymes from prokaryotic sources has been recognized and exploited in presentday molecular biology . In eukaryotes, so far only the viruses of green algae have been found to encode restriction endonucleases and DNA-methyltransferases . 47 In several instances, the loss of integrated viral DNA from the genomes of transformed cells has been reported with the maintenance or the concomitant abrogation of the transformed phenotype .""' Mechanism and frequency of these presumptive excision events are unknown . The number of anecdotal reports on the excision of foreign genomes suggests that they may not be infrequent . The ability to excise foreign integrated DNA could be considered the most effective defense mechanism of the host against this very incisive mutagenic event . It would be interesting to investigate how often adjacent cellular DNA sequences could be co-excised along with the foreign DNA . How is extraneous DNA recognized by the excision machinery as foreign DNA after its integration into the host genome? Could differences in methylation patterns between foreign and endogenous sequences play a role? How frequent are excision events in the host genome even in the absence of foreign DNA? Long-term silencing of foreign DNA by sequence-specific methylation Since there is no evidence for the occurrence of restriction endonucleases, DNAmethyltransferase(s) are apparently the only remnants of the prokaryotic restrictionmodification system in the higher eukaryotes . It is conceivable that only a few DNAmethyltransferases exist in mammalian cells, but this issue has not yet been resolved . The only cloned gene" for this enzyme from mouse cells seems to be a unique gene, and its DNA-methyltransferase domain exhibits a surprisingly high homology to a consensus sequence characteristic for prokaryotic DNA-methyltransferases . 52 The occasionally held notion that only one DNA-methyltransferase may exist in mammalian cells raises the difficult question of how one enzyme should account for both the maintenance and the de novo methylation of DNA . Since the two reactions differ very significantly in their signal-recognition parts, hemimethylated versus unmethylated 5'CG-3' dinucleotides, respectively, it is difficult to envisage how one enzyme, without modifications or auxiliary factors, would be sufficient to catalyse both types of DNA modifications . Additional DNA methyltransferases are likely to exist . A gradual increase of DNA methylation in the integrated foreign genome has been observed after both the explantation of adenovirus-induced tumor cells into culture and the transfection of cells in culture with the adenoviral genome, and upon co-



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selection of viral DNA-transformed cells for the persistence of the co-transfected gene for neomycin phosphotransferase (S . T . Tjia, G . Meyer zu Altenschildesche, and W . Doerfler, unpublished data) . The de novo methylation of the foreign genome is initiated at sites close to the center of the co-linearly integrated viral DNA and does not directly spread into the foreign genome from sequences in the abutting host genome . The apparently selective sites of initiation of DNA methylation have been identified in several independently established cell lines ." We do not know yet whether a specific DNA sequence, structure or chromatin arrangement in the integrated viral genome or its specific distance from a point of reference in the host genome can serve as a potential signal to target the de novo methylation machinery . It has also been demonstrated by using the genomic sequencing technique that de novo patterns of DNA methylation can be generated by the spreading of methylation from a methylated 5'-CG-3' dinucleotide to non-methylated neighboring 5'-CG-3' dinucleotides . 3 ' .32 This spreading reaction of DNA methylation can be diverted by the presence of proteins bound to specific DNA sequences, but eventually affects these interactions and then involves even those 5'-CG-3' sequences previously seemingly protected from the spreading of the de novo methylation reaction . 32 Somehow, this spreading of DNA methylation has to be limited . It will be one of the interesting challenges in work on DNA methylation to identify the signals that direct and regulate the mechanism of de novo methylation . There are now many examples of gene technology experiments in which foreign genes have been integrated genomically and expressed in eukaryotic cells, including transgenic animals and plants . In numerous instances, these genes have been rapidly or gradually inactivated and their sequences have been found extensively methylated . This mode of inactivation can be considered a further cellular defense mechanism against the realization of foreign gene expression . After the failure of previous lines of defense, i .e . the mechanisms of degradation or excretion of extraneous DNA taken up into the cell or of the excision of foreign DNA from the genomes of transformed cells, the integrated foreign DNA is permanently inactivated by selective DNA methylation . It is conceivable that integrated adenovirus genes, e .g . that had proved selectively useful for the transformed cells, might be exempted from this long-term inactivation process . Of course, foreign DNA excision and degradation would constitute the definite cure from foreign genes . The example of integrated adenovirus genomes in which some of the integrated genes remain active and non-methylated presents evidence for the selectivity of the inactivation process . Some of the early adenoviral gene products like those of the El region have transactivating properties 4o,53 and hence, the continued activity of these genes may provide selective advantages at least for cells propagated in culture . Presently existing patterns of DNA methylation- vestiges of evolution? Patterns of DNA methylation are cell type-specific and reflect in a complex way the specific functional requirements of different cell types . At least for humans, a high degree of inter-individual concordance at the nucleotide' and the kilobase pair levels' has been described . In some segments of the human genome, patterns of methylation may show individual differences . 54 It is not known whether equivalent genes or DNA segments are methylated in comparable patterns among different species . Are there differences in methylation patterns between unique and repetitive DNA sequences, between exons and introns? In the course of evolution, foreign DNA may have been frequently incorporated into pre-existing genomes and methylated in a certain pattern, e .g . as the consequence of the aforementioned defense mechanism . The patterns of DNA methylation, which



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exist in today's species, may be the vestiges of evolution and the remnants of multiple methylation, perhaps demethylation and mutation events . Of course, details of these processes can no longer be traced . Methylation patterns are associated with the longterm inactivation of DNA sequences whose function has not been required in, or would have been even harmful to, the specific cell type and organism . These methylation and inactivation steps have been different in each cell type dependent on the functional specificities of each cell type . Hence, it would be expected that the existing DNA methylation patterns are cell type-specific . This hypothesis may also explain the claimed discrepancy that not all inactive genes are heavily methylated . Clearly, a number of genes will be inactive in a certain cell type most of the time, but may have to be occasionally activated . In that case, a long-term mechanism of inactivation, as that mediated by DNA methylation, would be inappropriate, since there are other effective ways of keeping a gene silent . My hypotheses leave the question still unansweredhow the DNA-methyltransferase system of the host cell can distinguish between DNA sequences that have to be inactivated for a long time and have to be methylated, and those whose activity will occasionally be required and which must not be methylated? DNA-binding proteins may have the ability to influence the methylation of DNA positively or negatively . The possibility exists that a pattern of DNA methylation in a given species can be re-evaluated, perhaps even changed during (early) embryonic development . Demethylation and remethylation events may occur during that phase in an organism's life .

Viral oncogenicity and de novo methylation of DNA As a corollary to the hypotheses formulated, the question can be raised as to what extent the integration of foreign (viral) DNA in virus-transformed cells and the ensuing methylation of the viral genomes can affect the levels and the patterns of DNA methylation in the flanking host cellular DNA sequences . Both de novo methylation and loss of methylation have been observed for cellular DNA sequences abutting foreign (viral) genomes . Over what range are these alterations of methylation patterns progressing, and can these changes in DNA methylation account for effects on the transcriptional program of virus-induced tumor cells? In what way are these alterations responsible for the oncogenic phenotype of virus-transformed cells?

Possible consequences of de novo methylation of foreign DNA for gene therapy It is not yet known how frequently foreign DNA integrated into an existing mammalian genome can become de novo methylated and the genes in it inactivated . We have observed de novo methylation in adenovirus DNA integrated in cell culture, 22,26,27 and in transgenic mice ." If this mechanism proved a more general one, the question would arise of how long genes added to cells or organisms in the course of gene therapy would remain unmethylated and continue to be expressed . For this reason investigations on the mechanism and the regulation of de novo methylation also appear important for practical purposes .

Research in the author's laboratory has been supported by the Deutsche Forschungsgemeinschaft through SFB74-TP2, and through Forschergruppe 'Immundysregulation and maligne Lymphome' . The contributions of several pre- and postdoctoral students in my laboratory have been identified by the appropriate literature references . I am indebted to Petra Bohm for expert editorial work .



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References 1 . Kochanek S, Toth M, Dehmel A, Renz D, Doerfler W. Interindividual concordance of methylation profiles in human genes for tumor necrosis factors a and /3 . Proc Natl Acad Sci USA 1990 ; 87 : 8830-4 . 2 . Behn-Krappa A, Holker I, Sandaradura de Silva U, Doerfler W . Patterns of DNA methylation are indistinguishable in different individuals over a wide range of human DNA sequences . Genomics 1991 ; 11 : 1-7 . 3 . Wang RY-H, Zhang X-Y, Ehrlich M . A human DNA-binding protein is methylation-specific and sequence-specific . Nucleic Acids Res 1986 ; 14: 1599-614. 4 . Meehan RR, Lewis JD, McKay S, Kleiner EL, Bird AP . Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs . Cell 1989 ; 58 : 499-507 . 5 . Doerfler W . DNA methylation-A regulatory signal in eukaryotic gene expression . J Gen Virol 1981 ; 57 : 1-20. 6 . Doerfler W . DNA methylation and gene activity. Annu Rev Biochem 1983 ; 52 : 93-124 . 7 . Doerfler W . DNA methylation : Role in viral transformation and persistence . Adv Viral Oncol 1984 ; 4 : 217-47 . 8 . Doerfler W . Complexities in gene regulation by promoter methylation . Nucleic Acids Mol Biol 1989; 3 : 92-119 . 9 . Doerfler W. The significance of DNA methylation patterns : Promoter inhibition by sequence-specific methylation is one functional consequence . Philos Trans R Soc Lond (Biol) 1990 ; B326 : 253-65 . 10 . Doerfler W . Patterns of DNA methylation-evolutionary vestiges of foreign DNA inactivation as a host defense mechanism . A proposal . Biol Chem Hoppe-Seyler 1991 ; 372 : 557-564 . 11 . Doerfler W, Weisshaar B, Hoeveler A, Knebel D, Muller U, Dobrzanski P, Lichtenberg U, Achten S, Hermann R . Promoter inhibition by DNA methylation : a reversible signal . Gene 1988; 74 : 129-33 . 12 . Doerfler W, Toth M, Kochanek S, Achten S, Behn-Krappa A, Orend G . Eukaryotic DNA methylation : facts and problems . FEBS Lett 1990 ; 268 : 329-33 . 13 . Gunthert U, Schweiger M, Stupp M, Doerfler W . DNA methylation in adenovirus, adenovirustransformed cells, and host cells . Proc Natl Acad Sci USA 1976 ; 73 : 3923-7 . 14 . Wienhues U, Doerfler W . Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections . J Virol 1985 ; 56 : 320-4 . 15 . Vardimon L, Neumann R, Kuhlmann I, Sutter D, Doerfler W . DNA methylation and viral gene expression in adenovirus-transformed and -infected cells . Nucleic Acids Res 1980 ; 8 : 2461-73 . 16 . Doerfler W . Animal virus-host genome interactions . In : Fraenkel-Conrat H, Wagner RR, eds . Comprehensive Virology, Vol . 10 . New York, London : Plenum Press, 1977 ; 279-399 . 17 . Doerfler W, Gahlmann R, Stabel S et a/. On the mechanism of recombination between adenoviral and cellular DNAs : the structure of junction sites . Curr Top Microbiol Immunol 1983 ; 109 : 193-228 . 18 . Doerfler W. The fate of the DNA of adenovirus type 12 in baby hamster kidney cells . Proc Natl Acad Sci USA 1968 ; 60 : 636-643 . 19 . Doerfler W . Integration of the deoxyribonucleic acid of adenovirus type 12 into the deoxyribonucleic acid of baby hamster kidney cells . J Virol 1970 ; 6 : 652-66 . 20 . Jessberger R, Heuss D, Doerfler W . Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences . EMBO J 1989 ; 8 : 869-78 . 21 . Doerfler W . The abortive infection and malignant transformation by adenoviruses: Integration of viral DNA and control of viral gene expression by specific patterns of DNA methylation . Adv Virus Res 1991 ; 39 : 89-128 . 22 . Sutter D, Westphal M, Doerfler W . Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells . Cell 1978 ; 14 : 569-85 . 23 . Sutter D, Doerfler W . Methylation of integrated viral DNA sequences in hamster cells transformed by adenovirus 12 . Cold Spring Harbor Symp Quant Biol 1979 ; 44: 565-8 . 24 . Sutter D, Doerfler W. Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression . Proc Natl Acad Sci USA 1980; 77 : 253-6 . 25 . Kruczek I, Doerfler W . The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression . EMBO J 1982; 1 : 409-14 . 26 . Kuhlmann I, Doerfler W . Shifts in the extent and patterns of DNA methylation upon explantation and subcultivation of adenovirus type 12-induced hamster tumor cells . Virology 1982 ; 118 : 169-80 . 27 . Orend G, Kuhlmann I, Doerfler W . The spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells . J Virol 1991 ; 65 : 4301-8 . 28 . Lichtenberg U, Zock C, Doerfler W . Integration of foreign DNA into mammalian genome can be associated with hypomethylation at site of insertion . Virus Res 1988 ; 11 : 335-42. 29 . Doerfler W . Transformation of cells by adenoviruses : Less frequently discussed mechanisms . In : Doerfler W, Bohm P, eds . Molecular mechanisms in malignant transformation by DNA viruses . Weinheim : Verlag Chemie . In press . 30 . Lettmann C, Schmitz B, Doerfler W . Persistence or loss of preimposed methylation patterns and de novo methylation of foreign DNA integrated in transgenic mice . Nucleic Acids Res 1991 ; 19 : 71317137 . 31 . Toth M, Lichtenberg U, Doerfler W. Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns . Proc Natl Acad Sci USA 1989 ; 86 :3728-32 .



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32 . Toth M, Muller U, Doerfler W . Establishment of de novo DNA methylation patterns . Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter . J Mol Biol 1990; 214 : 673-83 . 33 . Vardimon L, Kressmann A, Cedar H, Maechler M, Doerfler W . Expression of a cloned adenovirus gene is inhibited by in vitro methylation . Proc Natl Acad Sci USA 1982 ; 79 : 1073-7 . 34 . Langner K-D, Vardimon L, Renz D, Doerfler W . DNA methylations of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2 . Proc Natl Acad Sci USA 1984 ; 81 : 2950-4 . 35 . Langner K-D, Weyer U, Doerfler W . Trans effect of the El region of adenoviruses on the expression of a prokaryotic gene in mammalian cells : resistance to 5'-CCGG-3' methylation . Proc Natl Acad Sci USA 1986 ;83 :1598-602 . 36 . Muller U, Doerfler W . Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells : gene expression and stability of methylation patterns . J Virol 1987 ; 61 : 3710-20 . 37 . Kruczek I, Doerfler W . Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters : effect of promoter methylation on gene expression . Proc Natl Acad Sci USA 1983; 80 : 7586-90 . 38 . Dobrzanski P, Hoeveler A, Doerfler W . Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free transcription system . J Virol 1988; 62 : 3941-6 . 39 . Weisshaar B, Langner K-D, Juttermann R, Muller U, Zock C, Klimkait T, Doerfler W . Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13S) functions . J Mol Biol 1988; 202 : 255-70 . 40. Flint J, Shenk T. Adenovirus Ell A protein paradigm viral transactivator . Annu Rev Genet 1989 ; 23 : 141-61 . 41 . Thompson JP, Granoff A, Willis DB . Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein . Proc Natl Acad Sci USA 1986 ; 83 : 7688-92 . 42 . Knebel-Morsdorf D, Achten S, Langer K-D, Ruger R, Fleckenstein B, Doerfler W . Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 DNA by a strong enhancer of human cytomegalovirus . Virology 1988 ; 166 : 166-74 . 43 . Boyes J, Bird A . DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein . Cell 1991 ; 64 : 1123-34 . 44 . Hoeveler A, Doerfler W . Specific factors binding to the E2A late promoter region of adenovirus type 2 DNA : no apparent effects of 5'-CCGG-3' methylation . DNA 1987 ; 6 : 449-60 . 45 . Hermann R, Doerfler W. Interference with protein binding at AP2 sites by sequence-specific methylation in the late E2A promoter of adenovirus type 2 DNA . FEBS Lett 1991 ; 281 : 191-5 . 46 . Hermann R, Hoeveler A, Doerfler W . Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding . J Mol Biol 1989 ; 210 : 411-15 . 47 . Van Etten JL, Burbank DE, Schuster AM, Meints RH . Lytic viruses infecting a Chlorella-like alga . Virology 1985 ; 140 : 135-43 . 48 . Groneberg J, Sutter D, Soboll H, Doerfler W . Morphological revertants of adenovirus type 12transformed hamster cells . J Gen Virol 1978 ; 40 : 635-45 . 49 . Botchan M, Stringer J, Mitchison T, Sambrook J . Integration and excision of SV40 DNA from the chromosome of a transformed cell . Cell 1980 ; 20: 143-52 . 50 . Kuhlmann I, Achten S, Rudolph R, Doerfler W . Tumor induction by human adenovirus type 12 in hamsters : Loss of the viral genome from adenovirus type 12-induced tumor cells is compatible with tumor formation . EM BO J 1982; 1 : 79-86. 51 . Bestor T, Laudano A, Mattaliano R, Ingram V . Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells . The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases . J Mol Biol 1988 ; 203 : 971-83 . 52 . Walter J, Noyer-Weidner M, Trautner TA . The amino acid sequence of the CCGG recognizing DNA methyltransferase M .BsuFl : implications for the analysis of sequence recognition by cytosine DNA methyltransferases. EM BO J 1990; 9 : 1007-13 . 53 . Nevins JR . Regulation of early adenovirus gene expression . Microbiol Rev 1987 ; 51 : 419-30 . 54 . Silva AJ, White R . Inheritance of allelic blueprints for methylation patterns . Cell 1988; 54 : 145-52 .

DNA methylation: eukaryotic defense against the transcription of foreign genes?

Microbial Pathogenesis 1992 ; 12 : 1-8 Mini-review DNA methylation : eukaryotic defense against the transcription of foreign genes? Walter Doerfler I...
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