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The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.
FIG. 1. The Xenopus embryo genome does not undergo demeth- ylation after fertilization and during the very early cleavage stages. (A) Oocyte nucleus stained with an antibody against 5mC (green). (B) Nucleus from a one-cell-stage embryo stained for 5mC. (C) Nucleus from a two-cell-stage embryo stained for 5mC. (D) During the cleavage stages (stages 2â8), the chromosomes of the embryo replicate and segregate as independent units (karyomeres) without forming a typical interphase nucleus. Karyomeres from 16-cell embryos also stained well with the antibody against 5mC. (E) Nucleus from a stage 12.5 gastrula embryo stained for 5mC. Note different organization of chromatin clusters compared to (AâC). (F) Nucleus, stained with the antibody against 5mC, from stage 7.5â 8 embryo depleted of xDnmt1 by injection of anti-sense RNA at the two-cell stage. (GâL) Dapi staining of the same nuclei as in (AâF).
FIG 2. Demethylation activity is absent from Xenopus egg cyto- plasm. (A) Sperm nucleus stained with the antibody against 5mC. (B) Sperm nucleus incubated in an interphase egg extract for 45 min still stains well with the 5mC antibody. (C) Sperm nucleus after 90 min of incubation in the extract (corresponding to the time of the first cleavage of the zygote) has the same intensity of 5mC staining as the nuclei in (A) and (B). Note the progressive swelling of the sperm nuclei that reflects chromatin decondensation induced by the egg extract. (DâF) Double fluorescence of the same sperm nuclei as in (AâC), in blue for Dapi and in green for 5mC antibody. (G) Dot blot immunodetection of 5 methyl-cytosine in DNA from oocyte (Oo), blastulae stage 4 (B4), 7 (B7), and stage 12 gastrula (G) Xenopus embryos (upper panel left, in vivo). The amount of 5mC (5 methyl-cytosine) detected by the antibody progressively decreases toward gastrulation (compared to the hybridization signal for total DNA in the right side of the panel). There is no detectable change in the 5mC content of the oocyte and sperm nuclei incubated in interphase egg extract for 0, 45, and 90 min (lower left panel, in vitro). On the right side of the panel, the total DNA in the same spots (as on the left) was detected by DNA hybridization with the Satellite1 probe.
FIG. 6. Developmentally programmed changes in methylation of Xbra and cardiac Actin promoters are disrupted in maternal xDnmt1-depleted embryos. (A) Diagram indicating the HpaII sites of the Xbra, cardiac Actin, and mitochondrial ND1 regions that were analyzed by Southern blotting of genomic DNA from staged embryos. The broken line bars indicate the location of hybridization probes used in these experiments. DNA was isolated from staged wild-type (WT) and maternally xDnmt1-depleted [Dnmt1(ô°…)] staged embryos and digested with either HpaII restriction enzyme, methylation sensitive at CmCGG, or MspI, methylation insensitive at the same sequence, as a control. (B) A Southern blot hybridized with a 1.5-kb probe derived from the Xbra promoter. Note that the promoter is completely hypomethylated in xDnmt1-depleted blastulae compared to the WT but undergoes abnormal de novo methylation in xDnmt1(ô°…) gastrulae. (C) The same blot was rehybridized with a 2-kb cardiac Actin promoter probe. This promoter region fails to be methylated de novo in xDnmt1(ô°…) gastrulae as compared to the WT, which suggests that the de novo methylation signal is disrupted in xDnmt1-depleted embryos. (D) The blot was also rehybridized with a mitochondrial DNA probe (ND1) as a control for complete restriction enzyme digestion. Mitochondrial DNA is never methylated and is equally digested by both HpaII and MspI.
Xenopus oocytenucleus stained with an antibody against 5-Methylcytosine (5mC; 5-Methylcytosine AB1), in green.
