Mammalian genomes are compartmentalized into dense inactive chromatin that is hypermethylated and active open chromatin that is hypomethylated. It is generally accepted that this bimodal pattern of methylation is established during development and is then faithfully inherited through subsequent cell divisions by a maintenance DNA methyltransferase (DNMT1). The pattern of methylation is believed to direct local histone acetylation states. In contrast to this well accepted consensus, we show here

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... using a transient transfection model that an active demethylase is involved in shaping patterns of methylation in somatic cells. Demethylase activity is directed by the state of histone acetylation, and therefore, the resulting methylation pattern is determined by local histone acetylation states contrary to the accepted model. Our data support a new model suggesting that the pattern of methylation is maintained by a dynamic balance of methylation and demethylation activities and the local state of histone acetylation. This provides a simple mechanism for explaining why active genes are not methylated. A hallmark of mammalian genomes is the compartmentalization of the genome into dense inactive chromatin that is hypermethylated and active, open chromatin that is hypomethylated (1). However, the mechanism responsible for establishing this tight relationship remains unclear. The accepted model is that a sequence of methylation and demethylation events fashion the methylation pattern during development, but it is then faithfully inherited by a semiconservative DNA methyltransferase, DNMT1 1 (2). Methylation of newly synthesized DNA is exclusively determined by the state of methylation of the parental strand. The pattern of methylation is therefore believed to be fixed in somatic cells. Based on the assumption that DNA conserves its pattern of methylation in somatic cells, numerous experiments used transiently transfected methylated DNA to study the effects of DNA methylation on gene expression. In many of these studies the state of methylation of these ectopically methylated genes following transfection was not determined assuming that the pattern of methylation of the transfected gene did not change. Methylated DNA is associated with methyl CpG binding proteins, such as MeCP2, which reside in a complex with histone deacetylase activity (3). The current model is therefore that the pattern of methylation dictates the state of histone acetylation and chromatin configuration (4). This attractive model explains the compartmentalization of the genome and its inheritance in somatic cells, but it can not explain how genes are demethylated upon their activation. An alternative and opposite interpretation of the tight correlation between histone acetylation is that active chromatin causes demethylation of associated sequences. Such a model can explain why active genes are not methylated and how their unmethylated state is maintained through cell division. This hypothesis that active chromatin can cause demethylation is supported by previous data. Treatment of mammalian cells with general histone deacetylase inhibitors can cause global demethylation of human Epstein-Barr virus producer cell lines' genomes (5). Similarly in Neurospora the deacetylase inhibitor TSA causes selective demethylation (6). Recent data support the claim that inhibition of histone deacetylation can cause selective demethylation of some genes such as the IgfII receptor (7) but not certain tumor suppressor genes (8). Whereas this data shows that activation of genes by histone deacetylase inhibitors can lead toward loss of methylation, the mechanism is unclear. This loss of methylation might either be caused by inhibition of the maintenance DNA methyltransferase during replication, by site-specific proteins, by triggering site-specific repair activity, or by active site-specific or general demethylation. In this report we use a transient transfection approach to directly measure demethylation activity in human cells (HEK 293) and show that the state of methylation of DNA is not fixed in somatic cells but is dynamically modulated to correlate with the state of gene activity. This is accomplished by active demethylase activity that is directed by histone acetylation. These data provide a simple mechanism for explaining how active genes are demethylated and maintained in their unmethylated state. MATERIALS AND METHODS Cell Culture and CAT Assays-HEK 293 cells were plated at a density of 8 ϫ 10 4 /well in a six-well tissue culture dish and transiently transfected with 80 ng of plasmid DNA using the calcium phosphate precipitation method as described previously (9). Transfections were repeated a minimum of three times using different cultures of HEK 293 cells. CAT assays were performed in triplicate as described previously (9). Cell Culture and Flow Cytometry-HEK 293 cells were maintained as a monolayer in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% calf serum (Colorado Serum Co). To serum starve the cells, confluent HEK 293 cells were cultured in a medium containing 0.5% fetal calf serum for 72-h post-transfection. To determine the percentage of cells at different stages of the cell cycle, cells were stained with propidium iodide and the DNA content was measured by flow cytometry.