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Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice

Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA

View larger version (60K): [in a new window]   Fig. 1. Protein structure, sequence alignment and expression of Dnmt3L. (A) Schematic diagrams of mouse Dnmt3L (421 a.a.), Dnmt3a (908 a.a.), and Dnmt3b (859 a.a.) protein structures. The red block represents the PHD-like domain. The yellow bars represent the five highly conserved cytosine methyltransferase motifs in Dnmt3a and Dnmt3b. Dnmt3L contains motifs I, IV, VI, and part of IX, but not motif X. (B) Sequence alignment of the PHD-like domain. Conserved amino acid residues are highlighted in red while similar residues are highlighted in gray. The PHD-like domains are highly conserved among the Dnmt3 family members and the X-linked ATRX gene. (C) Sequence alignment of motifs I, IV, VI and IX. Note that Dnmt3L lacks the consensus amino acid residues PC in motif IV and ENV in motif VI, which form the catalytic center of cytosine methyltransferases. (D) A northern blot of total RNA (20 µg/lane) from undifferentiated ES cells (undiff) and differentiated embryoid bodies (diff) was hybridized with a Dnmt3L full-length cDNA probe. +, n, s and c represent wild-type and various Dnmt1 mutant alleles (Okano et al., 1998). The blot was rehybridized with a ß-actin probe as an RNA loading control. (E) Whole-mount in situ hybridization of E7.5 and E8.5 embryos using a Dnmt3L cDNA probe reveals high expression predominantly in the chorion. (F) X-gal staining of Dnmt3L+/– adult mice carrying the IRES-ßgeo marker driven by the endogenous Dnmt3L promoter shows Dnmt3L expression in the oocyte and in male germ cells in the seminiferous tubule. Scale bar: 40 µm.

View larger version (69K): [in a new window]   Fig. 2. Targeted disruption of Dnmt3L and analysis of DNA methylation in Dnmt3L–/– mutant ES cells. (A) Gene targeting at the Dnmt3L locus. The top line shows the wild-type genomic locus. The vertical bars represent the exons starting from the first coding exon 1 to exon 11. The targeting vectors were constructed by replacing exons 2-7, which encode the entire PHD domain and part of motif 1, either with an IRES-ßgeo or a PGK-hygromycin (Hyg) cassette. S, SacII; N, NsiI; RV, EcoRV; and B, BamHI. (B) Southern analysis of the genotype of mutant ES cell lines. Genomic DNA was digested with NsiI and hybridized with a 3' external probe. J1, the wild-type parental line; 277 and 280, Dnmt3L+/– lines; and 196, a Dnmt3L–/– line (ßgeo/hyg). (C-E) Genomic DNA from wild-type and mutant ES cell lines, as described in B, was digested with designated enzymes and hybridized to the MMLV, Igf2 DMR2, and the Igf2r region 2 probes. Two independent Dnmt3L–/– lines, 80 and 196, were used. No significant difference in DNA methylation was detected between wild-type and Dnmt3L–/– lines. m, maternal allele: p, paternal allele.

View larger version (110K): [in a new window]   Fig. 3. Gross morphology and histology of wild-type and Dnmt3L–/– testes. (A) Gross morphology of testes of wild-type (+/+) and Dnmt3L–/– (–/–) mice at 1 week and 8 weeks of age. (B-G) Testes from wild-type (B-D) and Dnmt3L–/– (E-G) mice at 1 week (B,E), 4 weeks (C,F), and 8 weeks after birth (D,G). The number of spermatogonia in 1-week old testes was not significantly different in wild-type and mutant mice. At 4 weeks of age, most seminiferous tubules in Dnmt3L–/– testes contained very few differentiated spermatocytes. At 8 weeks of age, Dnmt3L–/– testes contained almost no spermatids or spermatozoa, which are normally present in the lumen of wild-type seminiferous tubules. Scale bar: 100 µm.

View larger version (114K): [in a new window]   Fig. 4. Developmental defects of Dnmt3Lmat–/– embryos from Dnmt3L–/– mothers. (A) A wild-type E9.5 embryo, (B) a Dnmt3L–/– littermate, and (C) Dnmt3Lmat–/– embryos. Most Dnmt3Lmat–/– embryos (left, 85%) show mild defects such as a smaller brain, incomplete closure of the rostral end of the neural tube (arrow), and smaller branchial arches. Some Dnmt3Lmat–/– embryos (right, 15%) display severe developmental defects, including an open neural tube in the midbrain region (arrow), smaller forebrain, and incomplete turning.

View larger version (64K): [in a new window]   Fig. 5. Disruption of methylation and expression of maternally imprinted genes in Dnmt3Lmat–/– embryos. (A-D) Methylation of four maternally imprinted genes (Igf2r, Peg3, Snrpn and Peg1) were analyzed by either bisulfite sequencing or southern blot hybridization with genomic DNA isolated from E9.5 wild-type (+/+), Dnmt3L+/– (+/–), Dnmt3L–/– (–/–) and Dnmt3Lmat–/– (mat–/–) embryos. (A,B) Bisulfite sequencing analysis (methylation shown as black circles) shows that the Igf2r region 2 and Peg3 DMR are almost completely unmethylated in Dnmt3Lmat–/– embryos, whereas they are partially methylated in wild-type and Dnmt3L–/– embryos. (C,D) Southern blot analysis of the Snrpn DMR1 and the Peg1 DMR shows that the maternal alleles (m) of both genes are methylated in wild-type and Dnmt3L–/– embryos, but unmethylated in Dnmt3Lmat–/– embryos. (E,F) No change in methylation of paternally imprinted genes, Rasgrf1 and H19, was observed in Dnmt3Lmat–/– embryos. (G,H) Expression of four maternally imprinted genes (Igf2r, p57kip2, Peg1 and Snrpn) in E9.5 embryos was analyzed by either RT-PCR (G) or northern blot hybridization (H). RT-PCR analysis of expression of Igf2r and p57kip2 in two wild-type, two Dnmt3L–/–, and four Dnmt3Lmat–/– embryos showed that expression of Igf2r and p57kip2 was drastically reduced in all Dnmt3Lmat–/– embryos. Northern analysis of Peg1 and Snrpn expression with total RNA isolated from a pool of 5-6 E9.5 embryos for each genotype showed an increased expression (approximately twice the wild-type level) of Peg1 and Snrpn in Dnmt3Lmat–/– embryos as compared to the normal embryos. m, maternal allele; p, paternal allele.

View larger version (55K): [in a new window]   Fig. 6. Interaction between Dnmt3L and the Dnmt3 family methyltransferases. (A) COS cells transfected with GFP or GFP-3L alone (lanes 6 and 7) or along with myc-3a, myc-3b, or myc vector (lanes 1-5) were lysed and immunoprecipitated with anti-myc antibody. The bound proteins were separated on a 10% SDS-PAGE gel and immunoblotted with anti-GFP antibodies. The expression level of myc-3a and myc-3b in lanes 1 and 3 is shown by immunoblot using anti-myc antibody (lanes 8 and 9). The position of the GFP and GFP-3L is indicated. (B) NIH3T3 cells were transfected with myc-3L alone (not shown) or with myc-3L and GFP, GFP-3a, or GFP-3b. After 12 hours, the cells were fixed and immunostained with anti-c-myc antibodies followed by rhodamine-conjugated goat anti-mouse secondary antibodies. The myc-3L and GFP-3a proteins in transfected cells are shown as red and green fluorescence, respectively. The nuclei are stained with DAPI. The typical myc-3L localization patterns fall into four categories: nuclear, cytoplasmic, diffused and nuclear peripheral, and are shown in the top panels (red). In myc-3L alone or myc-3L and GFP, only cytoplasmic and diffused patterns were observed. In cells co-transfected with myc-3L and GFP-3a or GFP-3b, myc-3L is localized predominantly in the nuclei and colocalized with GFP-3a or GFP-3b. The percentage of cells expressing both myc-3L and GFP-3a or myc-3L and GFP-3b in each category is shown at the bottom. For myc-3L alone, 30 nuclei were scored and for other cotransfection experiments, 100-150 nuclei were scored.

View larger version (56K): [in a new window]   Fig. 7. Methylation of imprinted genes in embryos derived from a transplanted [Dnmt3a–/–, Dnmt3b+/–] ovary. (A-D) Methylation of four imprinted genes (Igf2r, Peg1, Peg3, and Snrpn) was analyzed either by Southern blot hybridization (A,B) or bisulfite sequencing (C,D) with DNA isolated from E9.5 wild-type (+/+), Dnmt3a+/– (+/–), and Dnmt3a–/– (–/–) embryos, and two embryos derived from the [Dnmt3a–/–, Dnmt3b+/–] oocytes (labeled as 1, 2). In A and B, DNA was digested with designated restriction enzymes and Southern blots were hybridized with probes of Igf2r region 2 (A) or Peg1 DMR (B). Bisulfite sequencing analysis of methylation (black circles) of Peg3 DMR and Snrpn DMR1 in wild type and embryos 1 and 2 derived from ovary transplantation (OVT) (C,D). The maternal alleles of Igf2r, Peg1, Peg3 and Snrpn were almost completely unmethylated in the two embryos derived from the [Dnmt3a–/–, Dnmt3b+/–] mother, but they were methylated in Dnmt3a+/– and Dnmt3a–/– embryos from Dnmt3a+/– mothers. (E,F) Methylation of H19 (E) and MMLV (F) remained unchanged. Methylation of the paternally imprinted H19 gene and non-imprinted endogenous MMLV DNA was unaffected in all embryos tested.

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