Review TRENDS in Biochemical Sciences Vol 31 No 2 February 2006 Genomic DNA methylation the mark and its mediators Robert J Klose and Adrian P Bird Wellcome Trust Centre for Cell Biology University of Edinburgh Michael Swann Building Mayfield Road Edinburgh EH9 3JR UK Methylation of DNA at position five of the cytosine ring occurs at most CpG dinucleotides in the mammalian genome and is essential for embryonic viability With several of the key proteins now known it has become possible to approach the biological significance of this epigenetic system through both biochemistry and genetics As a result advances have been made in our understanding of the mechanisms by which DNA methylation is targeted to specific regions of the genome and interpreted by methyl CpG binding proteins Recent studies have illuminated the role of DNA methylation in controlling gene expression and have strengthened its links with histone modification and chromatin remodelling Introduction DNA methylation is found in the genomes of diverse organisms including both prokaryotes and eukaryotes In prokaryotes DNA methylation occurs on both cytosine and adenine bases and encompasses part of the host restriction system reviewed in Ref 1 In multicellular eukaryotes however methylation seems to be confined to cytosine bases and is associated with a repressed chromatin state and inhibition of gene expression 2 DNA methylation is essential for viability in mice because targeted disruption of the DNA methyltransferase enzymes results in lethality 3 4 There are two general mechanisms by which DNA methylation inhibits gene expression first modification of cytosine bases can inhibit the association of some DNAbinding factors with their cognate DNA recognition sequences 5 and second proteins that recognize methyl CpG can elicit the repressive potential of methylated DNA 6 7 Methyl CpG binding proteins MBPs use transcriptional co repressor molecules to silence transcription and to modify surrounding chromatin providing a link between DNA methylation and chromatin remodelling and modification 8 13 In this review we focus on recent advances in our understanding of the mechanisms by which DNA methylation is targeted for transcriptional repression and the role of MBPs in interpreting the methyl CpG signal and silencing gene expression We emphasize examples from mammalian systems including studies on animal models because several recent reviews have covered topics of DNA methylation and silencing in plants and fungi 14 18 Corresponding author Bird A P A Bird ed ac uk Available online 5 January 2006 Targeting DNA methylation de novo DNA methyltransferases Mammalian cytosine DNA methyltransferase enzymes fit into two general classes based on their preferred DNA substrate The de novo methyltransferases DNMT3a and DNMT3b are mainly responsible for introducing cytosine methylation at previously unmethylated CpG sites whereas the maintenance methyltransferase DNMT1 copies pre existing methylation patterns onto the new DNA strand during DNA replication A fourth DNA methyltransferase DNMT2 shows weak DNA methyltransferase activity in vitro 19 but targeted deletion of the DNMT2 gene in embryonic stem cells causes no detectable effect on global DNA methylation suggesting that this enzyme has little involvement in setting DNA methylation patterns 20 DNMT3L is a DNMT related protein that does not contain intrinsic DNA methyltransferase activity but physically associates with DNMT3a and DNMT3b and modulates their catalytic activity 21 In combination these de novo and maintenance methyltransferases seem to constitute the core enzymatic components of the DNA methylation system in mammals Mechanisms of targeting de novo methylation Examples of global de novo methylation have been well documented during germ cell development and early embryogenesis when many DNA methylation marks are re established after phases of genome demethylation 22 It is difficult to study the mechanisms by which DNMT enzymes are recruited to the targeted DNA sequences at these times owing to the small amounts of biological material available for molecular and biochemical study Most current knowledge regarding targeting of DNA methylation de novo therefore comes from cell culture model systems These studies have suggested at least three possible means by which de novo methylation might be targeted first DNMT3 enzymes themselves might recognize DNA or chromatin via specific domains second DNMT3a and DNMT3b might be recruited through protein protein interactions with transcriptional repressors or other factors third the RNA mediated interference RNAi system might target de novo methylation to specific DNA sequences Figure 1 Here we consider evidence relating to these possibilities in turn In mouse cells DNMT3 enzymes partially localize to regions of pericentromeric heterochromatin Functional studies show that the conserved PWWP domain is www sciencedirect com 0968 0004 see front matter Q 2005 Elsevier Ltd All rights reserved doi 10 1016 j tibs 2005 12 008 90 Review TRENDS in Biochemical Sciences Vol 31 No 2 February 2006 a DNMT3A B PWWP DNMT3A B PWWP CH3 b DNMT DNMT TF CH3 TF c TGS DNMT CH3 CH3 TGS Ti BS Figure 1 Targeting de novo DNA methylation a The PWWP domain of the DNMT3a and DNMT3b enzymes is required for targeting DNA methyltransferase activity to regions of pericentromeric heterochromatin The PWWP domain might function as a DNA or chromatin targeting motif b Transcription factors TF have the capacity to interact with DNA methyltransferase enzymes DNMT and to recruit methyltransferase activity as a part of the molecular silencing repertoire used to shutdown gene expression c De novo DNA methylation might be targeted by transcriptional gene silencing TGS pathways that respond to RNAi signals The molecular mechanisms by which these silencing pathways are linked remain unknown required to target the catalytic activity to these regions of the genome Figure 1a The importance of this domain was highlighted by the discovery that a mutation in the PWWP domain of the human DNMT3b protein causes ICF syndrome a severe autosomal recessive disease in humans 23 The mutation abolishes normal chromatin binding by DNMT3b in tissue culture cells 24 and causes a reduction in DNA methylation of classical satellite 2 DNA in affected individuals 23 The crystal structure of the mouse Dnmt3b PWWP domain has been solved and in vitro evidence indicates that this domain might interact with