We propose an updated model for DNA methylation maintenance which takes account of the existing information and can provide a framework for future experimentation to define the mechanism of this key physiological and pathological process (). The localization of DNMT1 to the replication fork, the interaction of DNMT3A and 3B with nucleosomes bearing specific modifications, and cooperation between different methyltransferase enzymes are all needed for the maintenance of DNA methylation especially in repeats and imprinted genes6-8
. We propose that the bulk of DNA methylation in dividing cells is indeed maintained by Dnmt1 - the most abundant DNMT in the cell54
– in conjunction with UHRF1. With its marked preference for hemi-methylated sites DNMT1 is ideally suited for the maintenance of most DNA methylation. In this sense, the enzyme fulfills the original ideas put forward in the classic papers of 1975 (). It is important to emphasize that the main role of DNMT1 is in reading the DNA sequence and applying methyl groups opposite to newly replicated hemi-methylated CpG sites. In other words the enzyme “reads” the modifications on the DNA without regard for the chromatin configuration in which that particular piece of DNA is located.
Revised model for the maintenance of DNA methylation patterns
In addition to its action at the replication fork, there is also some evidence that DNMT1 is able to perform error correction. Tagging of newly replicated DNA with bromodeoxyuridine followed by methylation analysis shows that complete methylation of the CpG sites within the newly replicated DNA can be accomplished after the DNA has left the replication fork in mouse embryonic stem cells which have Dnmt1 as the sole known active DNA methyltransferase7
. Similar conclusions were reached by Schermelleh et al.39
using a different approach in mouse embryonic stem cells. Furthermore, in human cancer cell lines in which the PCNA binding site of DNMT1 has been disabled by genetic disruption, DNMT1 is able to complete the conversion of some of the hemi-methylated sites into fully methylated sites, as a function of time after the DNA has left the replication fork38,55
. UHRF1 might be involved in recruiting DNMT1 to hemi-methylated sites away from the replication fork but this has not yet been shown.
It seems possible that the high frequency of occurrence of 5-methylcytosines in methylated CpG islands and repeats might represent a challenge to the maintenance process because of the rapid generation of hemi-methylated sites during DNA synthesis. We argue that cooperativity between different DNMT enzymes might be required in mammalian cells to maintain DNA methylation of these densely methylated regions. Cooperativity might also be responsible for maintaining the methylation of repetitive elements such as Alus and LINES7
which, as mentioned previously, contain a high level of hemi-methylation in ES cells lacking Dnmt3a and 3b. We propose that Dnmt3a and 3b are associated with specific regions of DNA that need to be maintained as highly methylated through recruitment to specific nucleosomal contexts. For example, G9a which is associated with maintaining H3K9me3 and is frequently found at repeats might be responsible for recruiting DNMT3A and 3B to complete methylation after the DNA has left the replication fork. Indeed Schlesinger et al.56
have shown that DNMT3A is associated with methylated CpG islands. We have confirmed their results with regard to CpG islands and also with repeat elements7,37
. Our data also show the strong anchoring of both DNMT3A and DNMT3B but not DNMT1 to nucleosomes37
which presumably ensures their effective compartmentalization to methylated regions and does not allow for “free” enzymes to be present in the nucleus.
The DNMT3 enzymes anchored to nucleosomes containing methylated DNA37
do not “read” the methylation on the existing DNA sequence but are proposed rather to methylate sites missed by DNMT1 activity at the replication fork. In this sense the DNMT3 enzymes act like prokaryotic enzymes which are capable of maintaining methylation without regard for whether sites are hemi-methylated or completely unmethylated. If regions of DNA that are methylated remain associated with the DNMT3A and B after replication, this would ensure that methylation state of a region is maintained, rather than a specific methylation pattern. The evidence for maintenance of state rather than an exact CpG-by-CpG pattern comes from the observation that most methylated CpG islands do not have consistent DNA methylation patterns across all molecules7,36
and that unmethylated sites within methylated CpG islands have a high probability of undergoing de novo
. This is a key conceptual difference from the standard model of methylation maintenance; in our revised model which is similar to that proposed by Riggs and Xiong27
the methylation state in general is being copied rather than the specific patterns of methylated sites.
The key to the supportive roles for DNMT3A and 3B in DNA methylation maintenance is the continued association of the enzymes with their products after the enzymatic reaction has occurred. Indeed there are several examples with respect to chromatin modifying enzymes such as the EZH2 enzyme which remains associated with its H3-K27me3 product suggesting that this mechanism is quite common and may be responsible for the inheritance of epigenetic states57,58
The revised model lessens the earlier emphasis on “de novo
” and “maintenance” DNMTs with non-overlapping functions. It emphasizes the need for continual cooperativity between the DNMTs to maintain DNA methylation patterns after the DNA has left the replication fork. It also brings together the two separate pathways which have been suggested to be necessary for inheritance of DNA methylation and histone modification patterns respectively.59