DNA Methylation Machinery
Cellular Methylation Machinery and DNA Methylatransferases
DNA CELLULAR METHYLATION MACHINERY
DNA methylation patterns are known to be established by a complex interplay of at least three independent DNA methyltransferases: DNMT1, DNMT3A and DNMT3B. Homozygous loss of any of the three currently known mammalian DNMTs, which includes DNMT1, 3a, and 3b, has been described to be lethal in mice (Li et al., 1992).
DNMT1 is the most abundant methyltransferase in somatic cells (Robertson et al., 1999), localizes to replication foci (Leonhardt et al., 1992), has a 10-40-fold preference for hemimethylated DNA(Pradhan et al., 1999), and interacts with the proliferating cell nuclear antigen (PCNA) (Chuang et al., 1997). It is thought to be the enzyme responsible for copying methylation patterns after DNA replication, and therefore is often referred to as the ‘maintenance’ methyltransferase (Robertson and Wolffe, 2000A). DNMT1 is essential for proper embryonic development, imprinting and X-inactivation (Li et al., 1992; Li et al., 1993; Beard et al.,1995).
Both of the DNMT3 methyltransferases are required for the wave of de novo methylation that occurs in the genome following embryonic implantation, and for the de novo methylation of newly integrated retroviral sequences in mouse ES cells (Okano, et al., 1999). It is believed that both these enzymes have an equal preference for hemi- and unmethylated DNA substrates, and that is why they are referred to as ‘de novo methyltransferases’ (Okano et al., 1998).
Recent studies have depicted that all three enzymes possess both de novo and maintenance functions and that, at least in somatic cells, specific methyltransferases will be responsible for the methylation of certain genomic regions by their interactions with other nuclear proteins (Robertson and Wolffe, 2000A). Purification of a DNMT1 complex that contains the retinoblastoma (Rb) gene product, E2F1 and histone deacetylase 1 (HDAC1) (Robertson, et al., 2000); and yeast two-hybrid experiments that show that DNMT1 can form a complex with HDAC2 and the co-repressor proteins DMAP1 and tumour susceptibility gene 101 (TSG101) (Roundtree, et al., 2000).
In experimental systems, it has been demonstrated that methylation at promoters does not lead to silenced transcription until chromatin proteins are recruited to the region, which mediate the gene silencing (Kass et al., 1997). It is therefore believed, that methylation initiates the process that results in a loss of transcription, by recruiting chromatin remodeling proteins. The majority of our genome is normally packaged in a transcriptionally repressive chromatin state, of the type found in pericentromeric heterochromatin regions. This type of chromatin is heavily methylated, and is packaged into compacted nucleosomes that contain deacetylated histones, particularly deacetylated histone H3. These histones are extensively deacetylated through the action of histone deacetylases (HDACs). This deacetylated state maintains the nucleosomes in a tightly compacted, regularly spaced, and transcriptionally silent state (Murzina et al., 1999; Struhl, 1998). The histone mark of a methylated Lys9 residue on the tail of histone 3 (H3) is believed to target DNA methylation to the region, and along with deacetylated histones, demonstrates that this is transcriptionally repressive chromatin. H3 Lys9 methylation is maintained by a histone methyltranscferase (HMT) that is recruited by binding of the chromodomain protein HP1 to the methylated H3 Lys9 (Jones and Baylin, 2002).
DNA methylation is involved in the forming of the transcriptionally silent state of pericentromeric heterochromatin. Methyl-cytosine-binding proteins (MBPs) associate with methylated cytosines and also with numerous chromatin-remodelling complexes (Jones and Baylin, 2002). These MBPs reside in complexes that contain HDACs; for example, the methyl-binding proteins methyl-CpG-binding protein 2 (MECP2) and methyl-CpG-binding-domain proteins MBD1 and MBD2 have been found to associate with transcriptional co-repressors, such as SIN3, which are known to bind HDACs directly (Bird and Wolffe, 1999; Jones et al., 1998; Ng et al., 1999).
Only a small fraction of the genome is transcriptionally competent. Transcriptionally active chromatin, euchromatin, consists of unmethylated CpG sites, that are protected from DNMTs and repressive complexes containing HDACs by transcription activator complexes. The nucleosomes around the promoter are more widely spaced than in heterochromatin and contain heavily acetylated histones. The histone mark, methylation of Lys4 residue in histone 3, is found associated with transcriptionally permissive chromatin. Lys4 is methylated by a different histone methyltransferase (HMT), than Lys9 (Jones and Baylin, 2002).