DNA methylation can contribute to transcriptional silencing through several transcriptionally repressive complexes, which include methyl-CpG binding domain proteins (MBDs) and histone deacetylases (HDACs). We show here that the chief enzyme that maintains mammalian DNA methylation, DNMT1, can also establish a repressive transcription complex. The non-catalytic amino terminus of DNMT1 binds to HDAC2 and a new protein, DMAP1 (for DNMT1 associated protein), and can mediate transcriptional repression. DMAP1 has intrinsic transcription repressive activity, and binds to the transcriptional co-repressor TSG101. DMAP1 is targeted to replication foci through interaction with the far N terminus of DNMT1 throughout S phase, whereas HDAC2 joins DNMT1 and DMAP1 only during late S phase, providing a platform for how histones may become deacetylated in heterochromatin following replication. Thus, DNMT1 not only maintains DNA methylation, but also may directly target, in a heritable manner, transcriptionally repressive chromatin to the genome during DNA replication.

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis , worms, and mammals, but is absent from yeasts that have been examined. We identified and analyzed H3K27me3 in the filamentous fungus Neurospora crassa and in other Neurospora species. H3K27me3 covers 6.8% of the N. crassa genome, encompassing 223 domains, including 774 genes, all of which are transcriptionally silent. N. crassa H3K27me3-marked genes are less conserved than unmarked genes and only ∼35% of genes marked by H3K27me3 in N. crassa are also H3K27me3-marked in Neurospora discreta and Neurospora tetrasperma . We found that three components of the Neurospora Polycomb repressive complex 2 (PRC2)—[Su-(var)3–9; E(z); Trithorax] (SET)-7, embryonic ectoderm development (EED), and SU(Z)12 (suppressor of zeste12)—are required for H3K27me3, whereas the fourth component, Neurospora protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, caused by deletion of the gene encoding the catalytic PRC2 subunit, set-7 , resulted in up-regulation of 130 genes, including genes in both H3K27me3-marked and unmarked regions.

We demonstrate that the recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, like DNMT1, repress transcription in a methylation-independent manner. Dnmt3a and Dnmt3b repress transcription primarily through a plant homeodomain-like motif that is shared with the ATRX protein but is not present in DNMT1. Unlike DNMT1, which localizes to replication foci during S-phase in murine embryonic fibroblasts, Dnmt3a co-localizes with heterochromatin protein 1 ␣ (HP1␣) and methyl-CpG binding proteins throughout the cell cycle to late-replicating pericentromeric heterochromatin. In contrast to Dnmt3a, Dnmt3b remained diffuse in the nucleus of embryonic fibroblasts at all cell cycle stages. However, Dnmt3a and Dnmt3b co-localize to these pericentromeric heterochromatin regions in murine embryonic stem cells. This finding is important to the fact that mutations in DNMT3B are found in the developmental syndrome, ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies), which involves extensive loss of methylation from pericentromeric regions. The localization of Dnmt3a and Dnmt3b was unaffected in Dnmt1 null embryonic stem cells, which lose the majority of methylation at pericentromeric major satellite repeats, suggesting that these enzymes are not dependent upon preexisting methylation for their targeting. DNMT1 is then positioned to reestablish transcriptionally repressive chromatin as cells replicate, while Dnmt3a and Dnmt3b may help to establish such chromatin in late S-phase and maintain this repressive heterochromatin throughout the cell cycle in a developmentally and/or cell type manner.DNA methylation is essential for embryonic development and important to transcriptional repression of imprinted genes, genes on the inactive X chromosome, and critical genes aberrantly silenced in many human cancers (1, 2). This DNA modification is mediated by DNA methyltransferases (Dnmts) 1 . Three active Dnmts (Dnmt1, Dnmt3a, and Dnmt3b) are required for the establishment and maintenance of genomic methylation patterns and proper murine development (3, 4). Mutations in DNMT3B have been found to cause the developmental syndrome, ICF, which is characterized by a loss of methylation at pericentromeric heterochromatin (4 -6). Dnmt3a and Dnmt3b are thought to primarily methylate DNA de novo while Dnmt1 principally maintains heritable methylation patterns during DNA replication (4, 7). For this latter function, Dnmt1 has been shown to localize in the nucleus to DNA replication foci (7). More recently, DNMT1 was shown to possess transcriptional repression capabilities, independent of catalyzing DNA methylation, that were mediated partially through a direct interaction with the histone deacetylases, HDAC1 and HDAC2, and other co-repressors (8 -10). Furthermore, HDAC2 was found to co-localize with DNMT1 at replication foci only during the later stages of S-phase (9).In the present work, we wanted to determine whether Dnmt3a and Dnmt3b, like DNMT1, target to DNA replication foci and possess functions other...

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