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DNA methylation, gene regulation and imprinting
4
PL 4
DNA METHYLATION, GENE REGULATION AND IMPRINTING
W. DOERFLER, Institute of Genetics, University of Cologne, Cologne, F.R.G.
In analyzing the overall biological implications of DNA methylation, it has been useful to take a critical reductionist approach and decipher the effects on reactions that can be measured in a reliable way. Work on the inhibitory function of sequence-specific promoter methylations offers an opportunity to contribute to the understanding of gene regulation. It has been established by extensive studies in several eukaryotic systems that sequence-specific promoter methylation causes gene inactivation (for reviews i-5). The decisive site(s) are different for different promoters. We have tried to decipher this inhibitory mechanism in as much detail as possible. After having studied gene regulation in detail, one can begin to consider more complicated consequences of DNA methylation patterns, e.g., the involvement in genomic imprinting, development and differentiation or X chromosome inactivation. The study of these complex biological problems is based in part on the evidence for an inactivating function of sequence-specific methylations in eukaryotic gene expression. Patterns of methylation seem to be inherited in cell lines carried in culture and perhaps in organisms as well. The mode of inheritance is poorly understood. Many genomic segments in mammalian DNA will have to be investigated under a variety of conditions before valid generalized conclusions can be drawn. The type of inheritance of a given pattern of methylation may depend on the geography in the mammalian genome. One of the m o s t important unresolved problems in work on DNA methylation is generated, how it is maintained, and whether it is subject to fluctuations. In eukaryotes, the influence of DNA methylation on functions other than gene regulation has not yet been studied in detail. Considering the complexities in patterns of methylation in well studied segments of the mammalian chromosome, the suspicion arises that the observed patterns might be composed of several interdigitating patterns each of which might have a different function. In my laboratory, we have used the adenovirus EIA and late E2A promoters to demonstrate that sequence-specific methylations lead to the inhibition or inactivation of these promoters. For the late E2A promoter, inactivation by methylation at three 5'-CCGG-3' sequences has been shown in transient expression system (6, 7),after fixation in the host genome (8) or in an in vitro transcription system (9). Promoter inhibition by DNA methylation can be, at least partly, overcome by the EIA 289aa transactivating protein of adenoviruses (7, iO) or by a strong enhancer near the methylated promoter (ii). By using the genomic sequencing technique (12), we have recently determined the patterns of methylation in all CpG residues on an active or an inactive late E2A promoter in transformed hamster cells (13). Methylation of the two downstream 5'-CCGG-3' sequences at positions +6 and +24 of the late E2A promoter compromises the binding of specific proteins. The interference might account in some ways for the inhibitory effect of promoter methylation (14). Structural alterations of the promoter by methylation could also play a role. (Supported by the Deutsche Forschungsgemeinschaft through SFB74-CI and SFB274-TP2).
PL 4
DNA METHYLATION, GENE REGULATION AND IMPRINTING
W. DOERFLER, Institute of Genetics, University of Cologne, Cologne, F.R.G.
In analyzing the overall biological implications of DNA methylation, it has been useful to take a critical reductionist approach and decipher the effects on reactions that can be measured in a reliable way. Work on the inhibitory function of sequence-specific promoter methylations offers an opportunity to contribute to the understanding of gene regulation. It has been established by extensive studies in several eukaryotic systems that sequence-specific promoter methylation causes gene inactivation (for reviews i-5). The decisive site(s) are different for different promoters. We have tried to decipher this inhibitory mechanism in as much detail as possible. After having studied gene regulation in detail, one can begin to consider more complicated consequences of DNA methylation patterns, e.g., the involvement in genomic imprinting, development and differentiation or X chromosome inactivation. The study of these complex biological problems is based in part on the evidence for an inactivating function of sequence-specific methylations in eukaryotic gene expression. Patterns of methylation seem to be inherited in cell lines carried in culture and perhaps in organisms as well. The mode of inheritance is poorly understood. Many genomic segments in mammalian DNA will have to be investigated under a variety of conditions before valid generalized conclusions can be drawn. The type of inheritance of a given pattern of methylation may depend on the geography in the mammalian genome. One of the m o s t important unresolved problems in work on DNA methylation is generated, how it is maintained, and whether it is subject to fluctuations. In eukaryotes, the influence of DNA methylation on functions other than gene regulation has not yet been studied in detail. Considering the complexities in patterns of methylation in well studied segments of the mammalian chromosome, the suspicion arises that the observed patterns might be composed of several interdigitating patterns each of which might have a different function. In my laboratory, we have used the adenovirus EIA and late E2A promoters to demonstrate that sequence-specific methylations lead to the inhibition or inactivation of these promoters. For the late E2A promoter, inactivation by methylation at three 5'-CCGG-3' sequences has been shown in transient expression system (6, 7),after fixation in the host genome (8) or in an in vitro transcription system (9). Promoter inhibition by DNA methylation can be, at least partly, overcome by the EIA 289aa transactivating protein of adenoviruses (7, iO) or by a strong enhancer near the methylated promoter (ii). By using the genomic sequencing technique (12), we have recently determined the patterns of methylation in all CpG residues on an active or an inactive late E2A promoter in transformed hamster cells (13). Methylation of the two downstream 5'-CCGG-3' sequences at positions +6 and +24 of the late E2A promoter compromises the binding of specific proteins. The interference might account in some ways for the inhibitory effect of promoter methylation (14). Structural alterations of the promoter by methylation could also play a role. (Supported by the Deutsche Forschungsgemeinschaft through SFB74-CI and SFB274-TP2).