Role of De Novo DNA Methyltransferases in Initiation of Genomic Imprinting and X-Chromosome Inactivation

plays a key role in regulation of developmental gene expression, maintenance of genomic integrity, genomic imprinting, and X-chromosome inactivation (X-inactivation) in mammals. Methylation of mammalian genomic DNA occurs almost exclusively at the cytosine of CpG dinucleotides. The CpG methylation pattern of the mammalian genome is created and maintained by a combination of de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and a maintenance DNA methyltransferase Dnmt1. Targeted disruption of these DNA methyltransferase genes in mice results in embryonic or early postnatal lethality, indicating that they are essential for normal mammalian development (Li et al. 1992; Okano et al. 1999). Genomic imprinting and X-inactivation are the wellcharacterized, major epigenetic phenomena of mammals that regulate allelic expression of autosomal genes and Xlinked genes, respectively (Lyon 1961; Reik and Walter 2001). Both phenomena are known to be crucial for normal mammalian development. Imprinting is initiated during male and female gametogenesis, marking a subset of autosomal genes (up to a few hundred) in a sex-specific way (paternal and maternal imprinting). The imprinted genes show either paternal-specific or maternal-specific monoallelic expression in the offspring (Reik and Walter 2001). Thus imprinting is dependent on the sex of the parent from which the gene is derived, but not on the sex of the individual that carries the gene. By contrast, X-inactivation is a dosage compensation mechanism found only in females, which equalizes the X-linked gene dosage between males (with one X and one Y chromosome) and females (with two X chromosomes) (Lyon 1961). In the embryo proper (the epiblast lineages), X-inactivation is initiated during early development, leading to random inactivation of either the paternal or the maternal X chromosome. However, in the extraembryonic lineages (trophoblast and primitive endoderm derivatives) of mice, preferential inactivation of the paternal X chromosome occurs (Takagi and Sasaki 1975). Thus X-inactivation can be subject to genomic imprinting (imprinted X-inactivation). Like the imprinting of autosomes, the imprinting of X chromosome is thought to occur in the parental germ line. Previous studies with the mouse embryos and ES cells deficient for Dnmt1 showed that DNA methylation plays an essential role in the maintenance of genomic imprinting and X-inactivation in the embryo proper (Table 1) (Li et al. 1993; Beard et al. 1995; Panning and Jaenisch, 1996; Sado et al. 2000). By contrast, in the trophoblast, the role of DNA methylation seems more relaxed (Table 1) (Caspery et al. 1998; Tanaka et al. 1999; Sado et al. 2000). However, whether DNA methylation is involved in their initiation has not been addressed. If DNA methylation were to play a role in the initiation step, Dnmt3a or Dnmt3b (or both) should be the key players because these are the enzymes that establish new genomic methylation patterns (Okano et al. 1999). We therefore asked whether Dnmt3a and/or Dnmt3b is involved in the initiation of autosomal imprinting and X-inactivation using the cells and embryos deficient for these genes. The Cre-loxP conditional gene knockout system was particularly useful because of the early lethality of conventional Dnmt3a or Dnmt3b knockout mice (Okano et al. 1999). In this article, we summarize the results obtained from these experiments and discuss the role of de novo DNA methylation in the initiation of the two epigenetic phenomena.