Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.     

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.     

Molecular enzymology of the catalytic domains of the Dnmt3a and Dnmt3b DNA methyltransferases

The C-terminal domains of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b harbor all the conserved motifs characteristic for cytosine-C5 methyltransferases. Whereas the isolated catalytic domain of Dnmt1 is inactive, we show here that the C-terminal domains of Dnmt3a and Dnmt3b are catalytically active. Neither Dnmt3a nor Dnmt3b shows a significant preference for the satellite 2 sequence, although Dnmt3b is required for methylation of these regions in vivo. However, the catalytic domain of Dnmt3a methylates DNA in a distributive reaction, whereas Dnmt3b is processive, which accelerates methylation of macromolecular DNA in vitro. This property could make Dnmt3b a preferred enzyme for methylation at satellite 2 repeats, since they are highly CG-rich. We have also analyzed the catalytic activities of six different mutations found in ICF (immunodeficiency, centromeric instability, and facial abnormalities) patients in the catalytic domain of Dnmt3b. Five of them display catalytic activities reduced by 10-50-fold; one mutant was inactive in our assay (residual activity <1%). These results confirm that a reduced catalytic activity of Dnm3b causes ICF. However, the mutations in general do not completely abrogate catalytic activity. This finding may explain why ICF patients are viable, whereas nmt3b knock-out mice die during embryogenesis.     

DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo

The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.     

CD44 alternative splicing senses intragenic DNA methylation in tumors via direct and indirect mechanisms

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.     

Exogenous expression of mouse Dnmt3 induces apoptosis in Xenopus early embryos

Mouse DNA (cytosine-5) methyltransferases Dnmt3a and Dnmt3b are expected to be de novo-type DNA methyltransferases. In the present study, we found that exogenously expressed mouse Dnmt3a or Dnmt3b induced abnormal cell clusters at the gastrulation stage in Xenopus embryos. The abnormal cells were judged to be apoptotic from the positive staining with the TdT dUTP nucleotide end-labeling method and the rescue by hBcl-x(L), a Bcl-2 homologue. On the other hand, neither bacterial DNA (cytosine-5) methyltransferase nor Dnmt3b3, one of the three isoforms of Dnmt3b that has no DNA methylation activity, induced apoptosis. In addition, mutant Dnmt3a and the other two Dnmt3b isoforms, Dnmt3b1 and Dnmt3b2, which have no DNA methylation activity due to a change of the cysteine residue in the catalytic center to an alanine residue, retained the ability to induce apoptosis. This indicates that the apoptosis was not induced by DNA methylation activity. The domain of Dnmt3b1 (3b2) responsible for the apoptosis is the catalytic domain in the carboxyl-terminal half.     

Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L

The DNMT3-like protein, DNMT3L, is required for germ line DNA methylation, although it is inactive as a DNA methyltransferase per se. Previous studies have shown that DNMT3L physically associates with the active de novo DNA methyltransferases, DNMT3A and DNMT3B, and stimulates their catalytic activities in a cell culture system. However, the mechanism by which DNMT3L stimulates de novo methylation remains unclear. Here, we have purified the full-length human DNMT3A2 and DNMT3L proteins and determined unique conditions that allow for the proper reconstitution of the stimulation of DNMT3A2 de novo methyltransferase activity by DNMT3L. These conditions include the use of buffers resembling physiological conditions and the preincubation of the two proteins. Under these conditions, maximal stimulation is reached at equimolar amounts of DNMT3L and DNMT3A2 proteins, and the catalytic efficiency of DNMT3A2 is increased up to 20-fold. Biochemical analysis revealed that whereas DNMT3L on its own does not significantly bind to the methyl group donor, S-adenosyl-L-methionine (SAM), it strongly increases the binding of SAM to DNMT3A2. DNA binding, on the contrary, was not appreciably improved. Analysis of DNA methyltransferase complexes in solution using size exclusion chromatography revealed that DNMT3A2 forms large structures of heterogeneous sizes, whereas DNMT3L appears as a monomer. Binding of DNMT3L to DNMT3A2 promotes a dramatic reorganization of DNMT3A2 subunits and leads to the formation of specific complexes with enhanced DNA methyltransferase activity and increased SAM binding.     

Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b

We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or “maintenance,” of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.     

In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b

The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3' long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.     

Ectopic expression of DNA methyltransferases DNMT3A2 and DNMT3L leads to aberrant hypermethylation and postnatal lethality in mice

DNA methylation is generally known to inactivate gene expression. The DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, catalyze somatic cell lineage‐specific DNA methylation, while DNMT3A and DNMT3L catalyze germ cell lineage‐specific DNA methylation. How such lineage‐ and gene‐specific DNA methylation patterns are created remains to be elucidated. To better understand the regulatory mechanisms underlying DNA methylation, we generated transgenic mice that constitutively expressed DNMT3A and DNMT3L, and analyzed DNA methylation, gene expression, and their subsequent impact on ontogeny. All transgenic mice were born normally but died within 20 weeks accompanied with cardiac hypertrophy. Several genes were repressed in the hearts of transgenic mice compared with those in wild‐type mice. CpG islands of these downregulated genes were highly methylated in the transgenic mice. This abnormal methylation occurred in the perinatal stage. Conversely, monoallelic DNA methylation at imprinted loci was faithfully maintained in all transgenic mice, except H19. Thus, the loci preferred by DNMT3A and DNMT3L differ between somatic and germ cell lineages.     

Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation

DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression through cellular development and differentiation. Here we describe the consequences of single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes, at the DNMT3A·DNMT3A homotetramer and DNMT3A·DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady-state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the highly processive wild-type enzyme. Our results are consistent with the bimodal distribution of DNA methylation in vivo and the loss of clustered methylation in AML patients. Tetramerization with the known interacting partner DNMT3L rescues processive catalysis, demonstrating that protein binding at the DNMT3A tetramer interface can modulate methylation patterning. Our results provide a structural mechanism for the regulation of DNMT3A activity and epigenetic imprinting.