Abstract
Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process.
Highlights
90% of all CpGs in the mammalian genome are methylated at the 5 position of the cytosine ring
In order to dissect the establishment of methylation in the upstream region of the gene, mouse embryonic stem (ES) cells were differentiated in vitro for nine days: LIF was removed from the medium on day 1 and, 3 days later, retinoic acid (RA) was added for up to 6 days
To assess the specific contribution of each Dnmt in the establishment of the methylation pattern, we repeated the in vitro differentiation process (Figure 1B) with ES cells that were null for Dnmt3a, Dnmt3b, both Dnmt3a and Dnmt3b, or Dnmt1. These cells have been reported to be defective in the differentiation process [36],we found that the silencing of the Oct-4 gene under the influence of retinoic acid in both Dnmt1 and Dnmt3a/b mutants followed kinetics that were indistinguishable from wildtype ES cells (Figure S3 B-C)
Summary
90% of all CpGs in the mammalian genome are methylated at the 5 position of the cytosine ring. Several key proteins that affect this epigenetic modification are known; most importantly the DNA methyltransferases, Dnmt, Dnmt3a and Dnmt3b. Dnmt is the ‘‘maintenance methyltransferase’’ that localizes to replication foci during S phase [7] and copies the DNA methylation pattern to the newly synthesized daughter strand. Further support to this view comes from in vitro demonstrations that Dnmt preferentially methylates hemimethylated DNA [8]. Dnmt3a and Dnmt3b, on the other hand, are de novo methyltransferases, responsible for the methylation of unmodified DNA. Knock-outs of other protein coding genes, including G9a and Lsh, reduce global DNA methylation levels [10,11]
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