DNA methylation is commonly associated with gene silencing, and a link between histone deacetylation and DNA methylation has been established. However, the transcriptional impact of the position and length of methylated zones relative to the promoter and the coding region of a gene remains quite unclear. This study investigates the impact of regional methylation on transcription and the relationship between DNA methylation and histone acetylation. Using patch-methylated stable episomes in human cells, we establish the pivotal importance of the location of DNA methylation in the regulation of transcription. We further demonstrate that the size of the methylated patch is not a key determinant for transcriptional suppression. The impact of DNA methylation on transcription is greater when it is in the transcription unit, and it is primarily a local effect. However, methylation outside of the transcription unit may potentiate the effect of methylation within the transcription unit. Acetylated histones are associated with unmethylated DNA and are nearly absent from methylated DNA regions. This association appears to be local and does not propagate along the DNA.It has been well established that DNA methylation can influence gene expression. In general, DNA methylation represses transcription, and loss of methylation is associated with gene activation (4). DNA methylation can directly interfere with transcription factor binding in some cases (6). In other cases, DNA methylation does not inhibit transcription factor binding directly, but transcriptional repression occurs nevertheless. In these cases, methyl-CpG binding proteins can mediate histone modification by recruiting histone deacetylases to methylated DNA (18,19,22). Although it is clear that this is an important mechanism in the silencing of genes, many aspects of this remain undefined. Specifically, the impact of the position and size of the methylated DNA segments on regional transcription has not been sufficiently characterized to permit inferences about the impact on transcription.With fungi, it has been shown that methylation impacts transcriptional elongation more than initiation (2,20). Consistent with this finding, we previously found that methylation of the coding region of a luciferase gene represses transcription more than does methylation of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter (11). Several studies using nonreplicating plasmids have suggested that the size of the methylated region plays a major role in transcriptional suppression (15) and that methylation adjacent to the promoter may inhibit transcription (12,14,18). Interpreting the results of these studies is difficult because the experimental designs included the coding region in the methylated patch. Therefore, the impact of the size and location of the methylated patch on transcription may be more complicated than what was concluded in these studies.This study was designed to examine the impact of the size and location of the methylated patch on transcription and to in...

It has recently been shown that in Xenopus, DNA demethylation at promoter regions may involve protein-DNA interactions, based on the specificity of the demethylated sites. Utilizing a stable episomal system in human cells, we recently mapped the sites and dissected the steps of demethylation at oriP sites bound by EBNA1 protein. Although it is clear that protein binding is required for demethylation of the oriP sites, it is uncertain whether this is a unique feature of the replication origin or whether it is a general phenomenon for all DNA sequences to which sequence-specific proteins are bound. In the present study, we utilize the well-defined Escherichia coli lac repressor/operator system in human cells to determine whether protein binding to methylated DNA, in a region that is neither a replication origin nor a promoter, can also lead to demethylation of the binding sites. We found that demethylation specified by protein binding is not unique to the replication origin or to the promoter. We also found that transcriptional activity does not influence demethylation of the lac operator. Isopropyl-␤-D-thiogalactopyranoside (IPTG), an inhibitor of the lac repressor, can prevent demethylation of the lac operator DNA sites and can modulate demethylation of the lac operator by affecting the binding affinity of the lac repressor. Using this system, a titration of protein binding can be done. This titration permits one to infer that protein binding site occupancy is the determinant of demethylation at DNA sites and permits a determination of how this process progresses over time.CpG methylation is tightly regulated during DNA replication and differentiation of somatic cells. The preexisting DNA methylation pattern is maintained by DNA methyltransferase 1 (DNMT1) during DNA replication (14). Changes in the basic methylation pattern occur throughout development through the dynamic processes of de novo methylation and demethylation. Two gene products, DNMT3A and DNMT3B, have been recently reported to have methyltransferase activity both in vitro and in vivo (11, 17). These de novo methyltransferases are believed to be involved in gene regulation, X-inactivation, genomic imprinting, and methylation of endogenous retroviruses and transposable elements (for a review, see reference 27). Several demethylases have been described previously (2,13,21,24). Unfortunately, the activities of some of these reported demethylases cannot be reproduced in other laboratories (16,22), and in one case they cannot be reproduced in the laboratory reporting the activity (20). It has been found that genes lose their CpG methylation within the promoter when they become activated, whereas genes acquire CpG methylation after they are no longer transcribed (for reviews, see references 4 and 18). Matsuo et al. (15) suggested that demethylation may involve protein-DNA interactions in Xenopus embryos, based on the specificity of the demethylated sites. We have further demonstrated in an episomal system that binding of proteins specifies sites of demet...

CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.CpG methylation is considered to be important for development, X chromosome inactivation, genomic imprinting, and suppression of transposons in mammals. The critical role played by the establishment and maintenance of DNA methylation has been demonstrated by the findings that homozygous disruption of any one of the three known methyltransferases in mice leads to lethality (23,28). It has also been shown that some methyl-CpG binding proteins associate with histone deacetylase and repress transcription (for a review, see reference 6). The importance of this has been exemplified by the recent identification that a human disease, Rett syndrome, is caused by mutations in one of the methyl-CpG binding proteins, MeCP2 (1). Recently, changes in DNA methylation patterns have also been implicated in carcinogenesis (for a review, see reference 18).Three vertebrate genes with known methyltransferase function have been identified, namely, Dnmt1, Dnmt3a, and Dnmt3b (4,27). Dnmt1 has been considered as a maintenance methyltransferase mostly based on its association with the replication foci (22) and its preference for hemimethylated DNA (29,30,37). It has been reported that Dnmt1 is capable of methylating fully unmethylated DNA as well as non-CpG sites in vitro (30,37). Dnmt1 has also been described to have no site preference other than CpGs (37). In Dnmt1 knockout mice, there is still a considerable a...