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

Abstract

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.

FIG. 1.

Inactivation of Dnmt3a and Dnmt3b results in progressive loss of DNA methylation in ES cells. (A) Genomic DNA from Dnmt3a^−/− Dnmt3b^−/− ES cells (7aabb and 10aabb) that had been grown in culture for 5 to 40 passages, as well as wild-type (J1) and Dnmt1 mutant (n/n and c/c) ES cells, was digested with HpaII and hybridized to probes for endogenous C-type retrovirus repeats (pMO), minor satellite repeats, and IAP repeats. As a control for complete digestion, DNA from J1 cells was digested with MspI. The Dnmt1ⁿ allele (the “n” stands for N-terminal disruption) is a partial loss-of-function mutation (27) and the Dnmt1^c allele (the “c” stands for disruption of the catalytic or C-terminal domain) is a null mutation (24). (B) Genomic DNA from J1, Dnmt3a^−/− (6aa), or Dnmt3b^−/− (8bb) ES cells that had been grown in culture for 5 to 25 passages, as well as 7aabb (P40), was digested with HpaII and hybridized to the pMO probe. (C) Lysates from the indicated ES cell lines were immunoblotted with anti-Dnmt1 and anti-tubulin antibodies.

FIG. 2.

Stable expression of Dnmt3a and Dnmt3b isoforms in late-passage 7aabb cells. (A) Schematic diagram of Dnmt3a and Dnmt3b isoforms. The conserved PWWP and PHD domains, the methyltransferase motifs (I, IV, VI, IX, and X), and the sites of alternative splicing are indicated (the C-terminal 45 amino acids of Dnmt3b5 are out of frame and shown as an open bar). The locations of the epitopes for the Dnmt3a and Dnmt3b antibodies are also shown. (B) cDNAs encoding Dnmt3a/3b isoforms were subcloned in an expression vector (schematically shown at the top), and these constructs were individually electroporated into late-passage (P70) 7aabb cells, which were subsequently selected in blasticidin-containing medium for 7 days. Blasticidin-resistant clones were analyzed with immunoblotting with anti-Dnmt3a (middle panel) or anti-Dnmt3b (bottom panel) antibodies. As a loading control, the same membranes were immunoblotted with anti-tubulin antibody.

FIG. 3.

Expression of Dnmt3a/3b proteins in 7aabb cells restores DNA methylation. (A to D) Methylation of repetitive sequences. Genomic DNA from the indicated ES cell lines was digested with HpaII (A to C) or MaeII (D) and hybridized to the indicated probes. DNA from J1 cells digested with MspI was used as a control for complete digestion. (E) Analysis of the methylation status of the major satellite repeating unit by bisulfite sequencing. Genomic DNA from J1 and 7aabb cells, as well as from stable cell lines expressing Dnmt3a, Dnmt3a2, Dnmt3b1, and Dnmt3b3, was analyzed. The methylation status of six CpG sites from 8 to 12 individual clones is shown schematically (black circles represent methylated sites), and the percentages of methylated CpG sites are indicated in parenthesis. (F to I) Methylation of unique genes. The genomic DNA samples described in panels A to D were digested with BamHI and HhaI (F and H), EcoRI and HpaII (G), or EcoRV and HhaI (I) and hybridized to probes corresponding to the 3′ region of β-globin (F), the 5′ region of Pgk-1 (G), an exon of Pgk-2 (H), or the 5′ region of Xist (I). DNA from J1 cells digested with BamHI alone (F and H) or EcoRI alone (G) was used as controls.

FIG. 4.

Expression of Dnmt3a and Dnmt3b proteins in 7aabb cells fails to restore maternal methylation imprints. The same DNA samples described in Fig. 3 were digested with SacI and HhaI (A), BamHI and HpaII (B), PvuII and HpaII (C and D), or XbaI and HhaI (E) and hybridized to probes corresponding to the 5′ upstream region of H19 (A), the DMR2 of Igf2 (B), region 2 of Igf2r (C), the DMR of Peg1 (D), or the DMR1 of Snrpn (E). As controls, DNA from J1 cells was digested with the corresponding enzymes without HhaI or HpaII. The fragments derived from the paternal (p) and maternal (m) alleles are indicated.

FIG. 5.

Dnmt3b6 has no enzymatic activity in vivo. (A) Strategy of targeted deletion of Dnmt3b exons 21 and 22. The top line shows the Dnmt3b genomic structure with exons represented by vertical bars. The targeting vector (second line) was constructed by replacing exons 21 and 22 with a PGK-puromycin cassette. A PGK-DTA cassette was introduced for negative selection to increase the targeting frequency. (B) Southern analysis of the genotype of ES cell lines. Genomic DNA was digested with EcoRV and hybridized to a 3′ external probe, as shown in (A). The 16-kb wild-type allele, the 5-kb Dnmt3b1 targeted allele, and the 14-kb Dnmt3b null allele (30) are indicated. (C) Lysates from the indicated cell lines were immunoblotted with anti-Dnmt3b (top), anti-Dnmt3a (middle), and anti-tubulin (bottom) antibodies. (D and E) Genomic DNA from the indicated ES cell lines was digested with HpaII and hybridized to probes for endogenous C-type retrovirus repeats (D) and minor satellite repeats (E).

FIG. 6.

Active Dnmt3a/3b isoforms rescue the capacity of late-passage 7aabb cells to form terotomas in nude mice. (A) The indicated ES cell lines were injected into nude mice subcutaneously on both sides (three to four mice for each cell line, 5 × 10⁵ cells per site), and the mice were examined for teratomas after 4 weeks. A typical representation of the size of the teratomas derived from each cell line is shown. (B) Histological sections of teratomas derived from J1, early-passage (P10) 7aabb, and Dnmt3a, Dnmt3a2, and Dnmt3b1 stable clones showing the presence of multiple types of differentiated cells.

FIG. 7.

Dnmt1 and Dnmt3 proteins function cooperatively in maintaining methylation patterns. (A) Dnmt1 or Dnmt3a was overexpressed in 7aabb (P70) or Dnmt1^−/− (c/c) ES cells as indicated and stable clones were examined for protein expression by immunoblotting with anti-Dnmt1 (top), anti-Dnmt3a (middle), and anti-tubulin (bottom) antibodies. (B and C) Genomic DNA from the indicated ES cell lines was analyzed for methylation of repetitive sequences (B) and unique genes (C) with the indicated probes.

FIG. 8.

Model for the distinct roles of Dnmt1 and Dnmt3a/3b in de novo and maintenance methylation. (Step 1) Dnmt3a and Dnmt3b establish new DNA methylation patterns by de novo methylation of symmetric CpG dinucleotides (lollipops). (Step 2) Upon DNA replication, the new synthesized DNA becomes hemimethylated at CpG sites. (Step 3) Dnmt1, which is localized to the replication complex (RC), restores full methylation by methylating hemimethylated DNA. However, some CpG sites are left untouched by Dnmt1 and remain hemimethylated. (Step 4) Dnmt3a and Dnmt3b, which may also localize to the RC, recognize unmethylated CpG sites and restore methylation via de novo methylation. In summary, Dnmt1 is the major maintenance methyltransferase, and it has little or no de novo methylation activity in vivo. Dnmt3a and Dnmt3b possess active de novo methyltransferase activity in vivo and are essential for the establishment and maintenance of DNA methylation patterns.