Evidence that protein binding specifies sites of DNA demethylation.

CpG methylation plays an important role in mammalian development (31; for a review, see reference 2), and it has been correlated with repression of gene expression (22; for reviews, see reference 4 and 33). Changes in the basic pattern of de novo methylation and demethylation occur throughout development. How DNA regions are targeted for de novo methylation or demethylation and how the de novo methylation and demethylation processes are mediated are not clear. It has been hypothesized that protein factors may protect CpG islands from methyltransferase (1). However, the data to support this has been indirect. In vivo footprinting and DNA methylation studies on the phosphoglycerate kinase 1 (PGK-1) gene indicated protein-DNA contact in the promoter region of this gene on the active X chromosome but not on the inactive X chromosome (29, 30). It has been proposed by these authors that unidentified protein factors may be involved in keeping specific regions methylation free. In a second system, studies with transgenic mice have indicated that DNA sequences corresponding to Sp1 sites play an important role in protecting the CpG island of the adenine phosphoribosyltransferase (APRT) gene from methyltransferase (3, 24). However, a recent study (26) demonstrated the methylation-free status of the APRT gene in Sp1 knockout mice, and the authors suggested that the methylation-free status may be maintained by the binding of other members, such as Sp3, of the Sp1 family to the Sp1 sites. Hence, the identity of protein factors that might maintain sites of demethylation in this system remains uncertain. Study of the α-actin gene in myoblasts by using transient, nonreplicating plasmids indicated that demethylation of one DNA strand at a specific site occurs within 2 h after DNA enters the cells (28). Although it was not demonstrated directly in their study, Paroush et al. (28) suggested that demethylation may involve protein-DNA interactions based on the specificity of the demethylated sites. Two possible mechanisms for demethylation have been proposed in the above studies. In the first, a passive mechanism, demethylation is due to the failure of remethylation by maintenance methyltransferase, and hence demethylation of both DNA strands should occur in 50% of the cells after two rounds of replication (32). At least four or five rounds of replication would be required to demethylate about 95% of the DNA on both strands by this mechanism. In the second mechanism, an active mechanism, activity of a demethylase that may require cis- and trans-acting factors (reviewed in reference 38) can lead to demethylation of both strands on all the DNA without any extensive DNA replication. The hemimethylated chicken vitellogenin gene becomes symmetrically demethylated with limited DNA replication at 24 h after hormone stimulation in chicken liver (34). Transient transfection of the α-actin promoter upstream of a reporter gene into a rat cell line indicated that demethylation is a two-step process with a hemimethylated intermediate (28). Studies on δ-crystallin demethylation suggested possible hemimethylated intermediates (10, 36). However, after several rounds of DNA replication, no hemimethylated DNA could be detected by a sensitive PCR assay in an experiment in which in vitro-methylated sequences were injected into mouse zygotes (21). These studies support the hypothesis that demethylation is a multistep process and that a passive mechanism alone without an active mechanism participating at least in some steps cannot achieve complete demethylation. Considering that demethylation of the two DNA strands may involve different (distinguishable consecutive) mechanisms, four possible mechanisms can be postulated for the demethylation of the two DNA strands (Fig. ​(Fig.1):1): (i) protein binding could recruit a demethylase to demethylate both strands (active-active mechanism); (ii) protein binding could interfere with remethylation after replication, leading to demethylation of both strands on half of the DNA molecules after two rounds of replication (passive-passive mechanism); (iii) protein binding could recruit demethylase to demethylate one DNA strand, and then the first round of replication would lead to demethylation of both strands on half of the molecules (active-passive mechanism); and (iv) protein binding could interfere with remethylation after replication, and then the resulting hemimethylated DNA would recruit a demethylase to demethylate the second DNA strand (passive-active mechanism). While any one of these conceivable pathways may turn out to be the predominant one, it is conceivable that more than one may function during the course of cell differentiation and development at different DNA sites in different cell types. FIG. 1 Possible mechanisms of demethylation at specific sites. The term “passive” is used to indicate that the specific protein, such as EBNA-1, binds to its recognition sites and blocks remethylation at these sites by the maintenance methyltransferase ... It is well documented that plasmids bearing the latent replication origin of the Epstein-Barr virus (EBV), oriP, can be maintained in human cells expressing the EBV nuclear antigen EBNA-1 (17, 35, 40, 41). We have developed a stable episomal system in which oriP is used to study the dynamics of CpG methylation over time courses of several months in human cells (13). In our system, the CpG methylation patterns on the plasmids generated in vitro by using either FnuDII, HhaI, HpaII, or SssI methylase are maintained for months after transfection into human cells. This indicates that the maintenance methyltransferase can efficiently remethylate the newly synthesized strand at positions opposite the existing sites of CpG methylation. However, we have observed a few specific preferential demethylation sites on the episome. Among these few sites, three are HpaII sites in the oriP region that become demethylated very quickly after transfection into human cells expressing EBNA-1. The demethylation of these HpaII sites appears to be either simultaneous or close to simultaneous. In the current study, the sites of demethylation in oriP were mapped by using both Southern blot analysis and bisulfite genomic sequencing methods. Furthermore, experiments were designed to explore the mechanism of demethylation in this region. We found that protein binding is required for demethylation of the oriP region. Replication alone does not lead to demethylation of the oriP without EBNA-1 binding. Furthermore, EBNA-1 binding alone also does not lead to demethylation of the oriP before replication. The HpaII sites within the EBNA-1 binding sites remained at least hemimethylated after one round of DNA replication. Moreover, the ability to identify molecules that were not replicated or were replicated once or twice in this system allows the analysis of the steps and mechanism of demethylation in the oriP region. The first-strand demethylation of the oriP region occurs by a passive mechanism, and the second-strand demethylation of these sites is probably processed through an active mechanism. This mechanism offers a logical interpretation of observations on demethylation events of endogenous genes.