Methylation During the Life Cycle

We present a model for the kinetics of methylation and demethylation of eukaryotic DNA; the model incorporates values for de novo methylation and the error rate of maintenance methylation. From the equations, an equilibrium is reached such that the proportion of sites which are newly methylated equals the proportion of sites which become demethylated in a cell generation. This equilibrium is empirically determined as the level of maintenance methylation. We then chose reasonable values for the parameters using maize and mice as model species. In general, if the genome is either hypermethylated or hypomethylated it will approach the equilibrium level of maintenance methylation asymptotically over time; events occurring just once per life cycle to suppress methylation can maintain a relatively hypomethylated state. Although the equations developed are used here as framework for evaluating events in the whole genome, they can also be used to evaluate the rates of methylation and demethylation in specific sites over time. I N prokaryotes it is well-established that DNA modification plays many roles. For example, methylation determines recognition of self us. non-self DNA through the action of methylation-sensitive restriction enzymes, is involved in the regulation of transposon activity and determines the strand specificity of mismatch repair (reviewed in MESSER and NOYER-WEIDNER 1988). Although the role of 5’ methylation of cytosine residues in eukaryotic DNA is not as well understood, methylation is intriguing because there are many examples of a negative correlation between methylation and gene expression (reviewed by CEDAR 1988). Cytosine methylation is not, however, present in all eukaryotes, thus this DNA modification cannot be universally required for gene regulation. Among those organisms with 5-methylcytosine, the methylation substrate sites also vary. In vertebrates, C residues in the dinucleotide CpC are the substrate; in higher plants C residues in both CpC and CpXpC, where X equals any nucleotide except G, are substrate sequences (GRUENBAUM et a l . 198 1). One interesting feature of DNA methylation in eukaryotes is its relative stability; that is, methylation pattern is a cell-heritable phenotype, a feature similar to aspects of cell differentiation (RAZIN 1984). Cell inheritance occurs because maintenance methylation enzymes utilize a template-the methylation pattern of an original DNA strand-to impose a symmetric pattern of methylation on a newly synthesized DNA strand (Figure 1). Equally important, however, is the of page charges. This article nust therefore be hereby marked “aduerhsernent” The publication costs of this article were partly defrayed hy the payment in accordance with 18 U.S.C. 5 1734 solely t o indicate this fact. Genetics 124: 429-437 (February, 1990) ease with which an existing methylation pattern can be lost. This process is termed demethylation even though the process is not enzymatic. Instead methylation patterns are lost by virtue of the failure of the maintenance system after DNA replication. If a cell were to fail totally in maintenance methylation, the effect on methylation levels would be rapid and dramatic during subsequent cell proliferation. After one round of DNA replication in the absence of methylation, chromosomes in each daughter cell would contain a template strand carrying the original methylation pattern and an unmethylated strand. Thus, all substrate sites will either be hemi-methylated or unmethylated. Every subsequent round of DNA replication without methylation will halve the frequency of template strands with an associated increase in completely unmethylated strands. With renewed methylation, maintenance methylation can act on chromosomes containing a template strand so that most hemi-methylated sites become homomethylated. Substrate sites on completely unmethylated chromosomes will, however, become methylated much more slowly because de novo methylation of unmethylated DNA is infrequent. In fact, methylation is estimated to occur 10 to 200 times more frequently on hemimethylated substrates than on unmethylated ones (RAZIN 1984). This illustration of the properties of maintenance methylation applies equally well to regions of chromosomes-such as genes-which are differentially demethylated and remethylated compared to surrounding DNA. As a consequence of the properties of the methylation system, the pattern and extent of methylation are 430 S. P. Otto and V. Walbot inherently dynamic. Methylation can be rapidly lost in the absence of maintenance methylation, while de novo methylation is only slowly imposed. Previous discussions of the dynamics of methylation have employed a qualitative approach and have been limited to modeling a few cell divisions. Often the focus has been a specific gene or transcribed sequences in a particular cell type: these sequences are typically hypomethylated compared to bulk DNA presumably because transcriptional factors prevent or slow maintenance methylation. In this paper we explore the kinetics of methylation and demethylation in the whole genome through numerical simulations which cover many cell generations. Of course, at a particular substrate site, methylation is a discrete event. At any substrate C residue, a methyl group is either present or absent; in a duplex at any particular site, the two strands are either unmethylated, hemi-methylated or fully methylated. Considering the population of all substrate sites within a cell, or the individual substrate sites within a population of cells, we can examine the proportion of methylated sites as a continuous function. MATERIALS AND METHODS Recursive model of methylation: We take as our population the group of cytosines which can be methylated (an equivalent population consists of one site observed in many independent cells). We assume that this population is very large so that random drift may be ignored. The cell cycle with respect to replication and methylation of cytosine sites is assumed to occur as in Figure 1. Because DNA modification occurs after eplication and because the newly produced daughter strands are initially unmethylated, all methylated sites are on the template strand immediately after replication. At the time of methylation, a proportion (a) of the hemi-methylated residues becomes homomethylated by maintenance methylation. Furthermore, a proportion (P) of the unmethylated sites becomes homomethylated by de novo methylation. These properties can be described by the following equations. Let X,, Yn, and Z, be the proportion of homomethylA) DNA Replication .€amhuw E i d m t i o n -