The finding of inverse correlations between promoter activity and extent of DNA methylation led to the concept that sequence-specific promoter methylations exert a regulatory function on gene activity. In order to provide more direct evidence for this interpretation, we devised experiments in which a number of promoter-indicator gene constructs were tested for their genetic activities in the unmethylated or in the methylated state at 48 h after transfection into mammalian cells in culture. In general, these data corroborated the earlier interpretation of promoter inactivation by promoter methylation, although this experimental approach could, of course, not help decide whether in an intact mammalian genome promoter methylation was the cause or consequence of promoter inactivation. The former possibility, however, remains the more likely explanation.
The E2A promoter of Ad2 DNA. In a first set of experiments, the oocyte system from Xenopus laevis was adapted to test unmethylated or methylated promoter-gene constructs for genetic activities. The cloned E2A region of Ad2 DNA was then 5´-CCGG-3´-methylated with the HpaII DNMT or was left unmethylated. Subsequently, either construct was microinjected into the nuclei of Xenopus laevis oocytes. The methylation status of these constructs was maintained in the oocyte nuclei. At 48 h after microinjection, the unmethylated construct was transcribed in the oocyte nuclei; the methylated construct was silenced . Transcription was initiated at the authentic E2A late promoter of Ad2 DNA. Control constructs carrying the unmethylated histone H2 gene were actively transcribed when co-injected with the methylated and silenced E2A construct. Hence, there was no evidence for possible unspecific inhibitory effects exerted by the in vitro premethylated construct. Modification of the E2A construct by the BsuRI (5´-GG*CC-3´) DNMT did not inactivate transcription . These data provided direct evidence for the notion that 5´-CG-3´ sequence-specific promoter methylation was involved in the silencing of eukaryotic genes. The system was further refined by separating the promoter of the E2A gene from its body and preparing both DNA fragments in quantitative amounts. We then 5´-CCGG-3´ methylated either the promoter or the gene body part of the constructs. Subsequently, the methylated promoter was re-ligated to the unmethylated body of the E2A gene and, conversely, the unmethylated promoter was reattached to the methylated E2A gene sequence. Upon microinjection into the nuclei of Xenopus laevis oocytes, only the construct, in which the promoter had been methylated, was inactivated. The construct with an unmethylated promoter but a methylated gene body was actively transcribed . We interpreted these data to demonstrate that sequence-specific promoter methylation led to gene inactivation.
The E1A promoter of Ad12 DNA. A similar set of experiments was performed with constructs that carried the chloramphenicol acetyltransferase (CAT) gene as an indicator for gene activity under the control of the E1A regulatory region of Ad12 DNA. Methylation of the two HpaII (5´-CCGG-3´) or of the seven HhaI (5´-GCGC-3´) sequences in this promoter inactivated the CAT gene or severely decreased its activity at 48 h after the transfection of these constructs into mouse Ltk- cells [87, 88]. Several additional sites in the promoter of the E1A gene of adenovirus type 12 were methylated, and the activity of the modified promoter was assessed with the CAT indicator gene. The C-residue methylation of two AluI sites (5´-AGCT-3´) downstream from the TATA box had no effect on promoter activity. However, when one EcoRI (5´-GAATTC-3´) sequence, 281 bp upstream, or one TaqI (5´-TCGA-3´) site downstream from the TATA signal in the promoter was deoxyadenosine methylated, the promoter became silent . Deoxyadenosine methylation of an MboI (5´-GATC-3´) sequence downstream of the TATA signal had no effect. Apparently, methylated nucleotides introduced at highly specific promoter locations can play an important role in the down-regulation of the Ad12 E1A promoter at least in transfection experiments. Since N6-mA is not known to occur in mammalian DNA, the effect of N6-mA on promoter activity has been unexpected. In an extension of this experimental approach, additional viral and non-viral eukaryotic promoters were tested for their sensitivity towards 5´-CG-3´ or 5´-CCGG-3´ methylation. The CAT or luciferase gene was used as activity indicator 24 h after the transfection into different human cell lines (HeLa, PA-1, 293). The methylation of all 5´-CG-3´ sequences by the SssI DNMT inactivated the E2A late promoter of Ad2 DNA, the human cytomegalovirus promoter, the TNFalpha promoter, the herpes simplex virus thymidine kinase promoter, and decreased the activity of the SV40 early promoter . In some experiments, HpaII methylation just led to a decrease in genetic activity of some of these constructs.
The L1140 promoter of frog virus FV3 DNA. The resistance of this promoter to complete 5´-CG-3´ methylation and its full activity in fish or mammalian cells in the completely methylated state has been described above [82, 83].
The p10 promoter of the AcNPV insect virus. A construct, which contained the promoter of the p10 gene of the insect virus Autographa californica nuclear polyhedrosis virus (AcNPV) and the CAT indicator gene, was active in AcNPV-infected Spodoptera frugiperda insect cells at 18 h after transfection of the construct. When the three 5´-CCGG-3´ (HpaII) sites in the promoter and its downstream region were methylated, the p10 promoter was silenced . Although insect cells may contain only minor amounts of m5C, the activity of an AcNPV insect virus promoter could be shown to be sensitive to sequence-specific methylation.
Human Alu sequences transcribed by RNA polymerase III. We have also demonstrated that the polymerase III transcription of Alu sequences associated with the human angiogenin, the tissue plasminogen activator (TPA), or the alpha1-globin gene is inhibited by 5´-CG-3´ methylation of these sequences . Their methylation also interferes with the binding of proteins to the B control region of these Alu sequences .
Bending of promoter DNA sequences due to methylation? The site-specific methylation in 5´-CCGG-3´ (HpaII), 5´-CGCG-3´ (FnuDII), or in 5´-CG-3´ (SssI) sequences of the E2A promoter, the polymerase III-transcribed VAI RNA gene of Ad2 DNA or of the human angiogenin gene-associated Alu sequence can alter the electrophoretic mobility of these DNA sequences in non-denaturing polyacrylamide gels. This finding indicates that the bending of the tested sequences might be altered by DNA methylation .
Reversal of promoter inactivation by methylation. An adenovirus E1A gene product or the strong enhancer of human cytomegalovirus (HCMV) can overcome the transcription-inactivating effect of promoter methylation. The removal of the methyl group from m5C in a methylated promoter in the absence of DNA replication seems to be a rare event. Hence, other mechanisms for transient reactivation of a permanently methylated promoter appear to be required. Of course, experimentally, the methylated E2A late promoter in the Ad2-transformed cell line HE3 can be demethylated and reactivated by growing the cells in culture in the presence of 50 µM 5-azacytidine (5-aza-C), an inhibitor of maintenance methylation . This approach provides support of principle but does not adequately mimic the situation in a biological system.
In human 293 cells, which carry the left terminus of Ad5 DNA chromosomally integrated and express the E1 region of Ad5 constitutively, the inactivating effect of 5´-CCGG-3´ methylation of an E2A promoter construct of Ad2 DNA is released or markedly decreased . We have also shown that the E1A gene encoding the 13S RNA and the 289 amino acid (aa) protein of Ad2, a well-known transactivator of genes [96, 97], is responsible for the reversal of the inactivating effect of E2A promoter methylation . It is unknown, by which mechanism the 289 aa E1A function is capable of effecting this reactivation. The methylated E2A promoter did not lose its 5´-CCGG-3´ methyl groups in the reactivation process at 48 h after transfection. Moreover, the authentic cap-site of this promoter was used in the transcription following reactivation [94, 98]. Similarly, the 5´-CCGG-3´ methylated E2A promoter of Ad2 DNA was active when the strong immediate early enhancer of HCMV DNA was inserted into the promoter-indicator gene construct in a position either immediately antecedent to the promoter or several thousand nucleotides remote from it . Transcription was initiated also at the authentic cap-site of the E2A gene, and 5´-CCGG-3´ methylation remained unaltered at least during the duration of the transient expression experiment.
Promoter methylation and protein binding. This topic has been extensively investigated in several laboratories. In the E2A promoter system of Ad2 DNA, the in vitro methylation of 5´-CCGG-3´ sequences at nucleotides +24, +6, and -215 relative to nucleotide +1, the site of transcriptional initiation, was demonstrated to lead to transcriptional inactivation in transient expression studies in Xenopus laevis oocytes [86, 95], in mammalian cells , after the genomic fixation of the promoter in mammalian cells , and in a cell-free transcription system using nuclear extracts from human HeLa cells . DNA fragments, 50 or 73 bp in length, comprising the +24 and +6 5´-CCGG-3´ sequences of the E2A promoter of Ad2 DNA in the unmethylated, methylated, or hemimethylated state were incubated with partly purified nuclear extracts from human HeLa cells. Protein binding to these DNA preparations was assessed by electrophoretic mobility shift assays (EMSAs). The formation of one of the observed DNA-protein complexes in this system was compromised when the construct was methylated or hemimethylated . The results of the necessary competition experiments confirmed the interpretation that specific promoter methylation interfered with the binding of nuclear proteins from human cells. There was evidence that the AP2 transcription factor was among the proteins sensitive to promoter methylation in this system .