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. 2011 Mar 1;108(9):3642-7.
doi: 10.1073/pnas.1014033108. Epub 2011 Feb 14.

Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine (V体育平台登录)

Khursheed Iqbal  1 , Seung-Gi Jin, Gerd P Pfeifer, Piroska E Szabó
Affiliations

Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine

"V体育ios版" Khursheed Iqbal et al. Proc Natl Acad Sci U S A. .

Abstract

Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal VSports手机版. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals. .

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The authors declare no conflict of interest.

Figures (VSports最新版本)

Fig. 1.
Fig. 1.
5hmC is present in the male pronucleus of mouse zygotes. (A) A mouse zygote was double-stained with anti-5hmC antibody (green) and anti-5mC antibody (red). The smaller maternal pronucleus is closer to the polar body (pb). A bright-field image is shown on the far left. (B) Additional zygotes were double-stained with anti-5hmC antibody (green) and anti-5mC antibody (red). Merged images are shown. (C) Zygotes obtained by in vitro fertilization were double-stained similarly. Two polyspermic zygotes (to the right) exhibit 5hmC staining in two paternal pronuclei. (D) 5mC and 5hmC staining reveal two separate chromosome sets at metaphase of zygote division. A confocal image is shown. (E) Individual chromosomes are largely stained for either 5mC (likely originated from the maternal pronucleus) or 5hmC (likely from the paternal pronucleus) at anaphase of zygote division. Two Z sections of the same zygote are shown.
Fig. 2.
Fig. 2.
5hmC and 5mC in early pronuclear stage zygotes. (A) Zygotes at pronuclear stages PN1, PN2, and PN3 were double-stained with anti-5hmC antibody (green) and anti-5mC antibody (red). Merged images are shown. (B) The levels of 5hmC and 5mC in paternal and maternal pronuclei were quantitated. The ratio of staining signal between the paternal and maternal pronucleus is plotted. The number of zgotes analyzed in PN1/PN2 (Early), in PN3 (Mid), and in PN4/PN5 (Late) are indicated with n values. The median value is indicated by a horizontal line and a number. The difference between each two datasets is statistically significant, as seen in the P values of t-tests.
Fig. 3.
Fig. 3.
5hmC and 5mC in early cleavage-stage embryos. (A) Two-cell stage embryos were double-stained with anti-5hmC antibody (green) and anti-5mC antibody (red). pb, polar body. A bright-field image is shown on the far left. (B) Two-cell–stage embryos double-stained with anti-5hmC antibody (green) and anti-5mC antibody (red). These images were obtained by confocal microscopy. (C) Confocal microscopy image of a two-cell (2c) stage embryo entering mitosis. The condensed chromosomes are labeled with anti-5mC antibody (red) and anti-5hmC antibody (green). (D) 5hmC and 5mC in four- (4c) and eight-cell (8c) –stage embryos. Four-cell (Upper Left) and eight-cell (remaining images) embryos were double-stained with anti-5hmC antibody (green) and anti-5mC antibody (red). A confocal image is shown in the upper right image.
Fig. 4.
Fig. 4.
Expression of Tet and Stella/Dppa3 genes in oocytes, zygotes, and early cleavage-stage embryos. RNA was isolated from oocytes, zygotes, two-, four-, and eight-cell–stage embryos. Real-time PCR was used to assess the expression of the three Tet genes and Stella/Dppa3. Data were normalized relative to expression of β-actin. N.D., no detectable signal in real-time PCR. Expression of Tet1 in the zygote and of Tet3 at the two-cell stage has a detectable signal, which is close to zero.
Fig. 5.
Fig. 5.
Sodium bisulfite sequencing of Line1, ETn, Mylc, and Acta1 sequences in sperm, oocytes, and zygotes. DNA was isolated from mouse oocytes, sperm, or zygotes (PN4–PN5) and subjected to sodium bisulfite conversion. (A) Line1 5′ end sequences were amplified, cloned, and sequenced. Open squares, unmethylated CpGs; black squares, methylated CpGs; gray squares, not analyzable/mutated CpG site. Each row represents an individual sequenced DNA strand. (B) ETn sequences were amplified, cloned, and sequenced. The sequences from zygotes represent the paternal allele distinguishable by a sequence polymorphism. (C) Acta1 sequences. (D) Myl3 sequences. The percentage of methylated CpGs is indicated.
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"V体育官网" References

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