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

Abstract

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 prolif...

DNA methylation profiling in human B cells reveals immune regulatory elements and epigenetic plasticity at Alu elements during B-cell activation

“…A substantial portion of the celltype-specific differential methylation of Alus documented here correlates with decreased expression of the de novo methyltransferase DNMT3A at the GC stage (Fig. 1C,D), consistent with a model delineated by Jones and colleagues (Jeong et al 2009). Diminished expression of DNMT3A, coupled with increased replicative demand could lead to replication-dependent loss of methylation at repetitive elements.…”

“…In agreement with genome-scale studies in other cell types (Stadler et al 2011;Bock et al 2012;Thurman et al 2012), differentially methylated regions (DMRs) in GC B cells were enriched for TF binding sites or regulatory elements. A second class of loss-of-methylation events localized to Alu elements, consistent with the preferential targeting of DNMT3 family de novo methyltransferases to repetitive elements in the genome ( Jeong et al 2009). The methylation dynamics at Alu elements suggests a regulatory role of the repetitive fraction of the genome in the GC transcriptional program and/or its associated chromatin architecture, and a potential contribution to genomic instability in GC B cells (Ranuncolo et al 2007).…”

Phospho-ΔNp63α/microRNA network modulates epigenetic regulatory enzymes in squamous cell carcinomas

“…DNMT1 preserves the methylation DNA patterns throughout each cell division, while DNMT3A and 3B transfer a methyl group to unmethylated DNA sequences. [52][53][54][55][56] Although DNMT3A and 3B are believed to play a role of de novo DNA methyltransferases in development, recent studies showed that both DNMT3A and DNMT3B could also serve as maintenance enzymes that are responsible for copying DNA methylation patterns to the daughter strands during DNA replication. [52][53][54][55][56] Therefore, DNA methylation mediated by a combined action of DNMT1, DNMT3A, and DNMT3B is essential for understanding the epigenetic mechanisms underlying cellular transformation.…”

“…[52][53][54][55][56] Although DNMT3A and 3B are believed to play a role of de novo DNA methyltransferases in development, recent studies showed that both DNMT3A and DNMT3B could also serve as maintenance enzymes that are responsible for copying DNA methylation patterns to the daughter strands during DNA replication. [52][53][54][55][56] Therefore, DNA methylation mediated by a combined action of DNMT1, DNMT3A, and DNMT3B is essential for understanding the epigenetic mechanisms underlying cellular transformation. [52][53][54][55][56] Our initial studies that employed the high-throughput DNA methylation chip arrays showed that many sequences were exclusively hypermethylated in SCC-11M cells upon cisplatin exposure, compared with SCC-11 cells treated with cisplatin (data not shown).…”

“…[52][53][54][55][56] Therefore, DNA methylation mediated by a combined action of DNMT1, DNMT3A, and DNMT3B is essential for understanding the epigenetic mechanisms underlying cellular transformation. [52][53][54][55][56] Our initial studies that employed the high-throughput DNA methylation chip arrays showed that many sequences were exclusively hypermethylated in SCC-11M cells upon cisplatin exposure, compared with SCC-11 cells treated with cisplatin (data not shown). Among these sequences, the DAPK1 promoter area was found starting at −1768 bp (Fig.…”