Selective Anchoring of DNA Methyltransferases 3A and 3B to Nucleosomes Containing Methylated DNA

Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.Proper DNA methylation patterns are essential for mammalian development and differentiation. More than three decades ago, de novo cytosine DNA methylation and its maintenance were proposed to exist in eukaryotic cells (29, 54); however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. DNA methyltransferases (DNMTs) DNMT1, DNMT3A, and DNMT3B primarily establish and maintain global DNA methylation patterns (39, 48). DNMT1 preferentially methylates hemimethylated DNA in vitro (7) and is tethered to replication foci during S phase (38). In contrast, DNMT3A and DNMT3B (DNMT3A/3B) have no preference for hemimethylated DNA (49) and are required for de novo methylation of genomic DNA (48). It has been thought that DNMT1 acts mainly as a “maintenance methyltransferase” during DNA synthesis and that DNMT3A and DNMT3B act as “de novo” enzymes. However, more recent studies indicate that DNMT1 may also be required for de novo methylation of genomic DNA (17, 30) and that DNMT3A/3B are also required for maintenance functions (11, 40, 55). Furthermore, the different DNMTs cooperate in maintaining the methylation of some regions of the genome, particularly repetitive elements (40, 53).Recruitment of individual DNMT enzymes to different regions of chromatin in vivo, particularly to gene regulatory regions, may require interaction with auxiliary factors (28, 36). DNMT1, which is diffusely localized throughout nuclei in non-S-phase cells (38), is targeted to replication foci by interacting with proliferating cell nuclear antigen (PCNA) (15) and also physically interacts with UHRF1 (ubiquitinlike, containing PHD and RING finger domains 1) that binds to hemimethylated DNA (3, 4, 8, 27, 62). DNMT3 enzymes are usually found localized to heterochromatin regions in most transient-expression assays (5, 12). As genomic DNA in chromatin is packaged into nucleosomes which might limit the accessibility of target sites to the enzymes, the interaction of DNMTs with nucleosomes in a chromatin context is important for the regulation of genomic methylation.Genetic and biochemical studies have provided many insights into the distinct and cooperative functions of the DNMT enzymes; however, few of these studies have addressed how they interact with chromatin in vivo. Recombinant DNMT1 and DNMT3 enzymes can methylate the CpG sites on nucleosomes assembled in vitro (26, 50, 56, 65). Recently DNMT3L has been found to connect DNMT3A2 to nucleosomes in embryonic stem cells (52). However, DNMT3L is expressed only during gametogenesis and embryonic stages (1, 9), suggesting that other mechanisms might be necessary for directing the enzyme to specific chromatin regions in somatic cells.In the present study, we investigated how different DNMT enzymes interact with chromatin at the nucleosomal level in somatic cell lines. Micrococcal nuclease (MNase) treatment of nuclei in a low-ionic-strength buffer digests nucleosomal linker DNA regions, thereby minimizing the disruption of protein complexes on the nucleosomes. We prepared nucleosomes from partial or maximum MNase-digested nuclei and resolved them on sucrose density gradients to analyze their interactions with chromatin proteins. The results indicate that while DNMT1 interacts primarily with linker DNA, DNMT3A/3B enzymes interact strongly with nucleosomes containing methylated repetitive elements and also containing methylated CpG islands (CGIs) and may not require additional proteins for this strong binding. These data are particularly intriguing in that they provide insights into the mechanisms of the interaction of DNMTs with chromatin and maintenance of DNA methylation in somatic cells.