A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found thatHpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is l10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days, about the time of X-chromosome inactivation of the inner cell mass.An assay based on the use of HpaII prior to the polymerase chain reaction (PCR) (20,22) allowed us to study DNA methylation in individual mouse embryos at the time of X-chromosome inactivation. We probed methylation in the CpG-rich island at the 5' end of the X-linked gene for phosphoglycerate kinase (Pgk-J), at a HpaII site (H-7) showing female-specific methylation in adult somatic tissues (Fig. 1). We found that site H-7 is not significantly methylated in male embryos but that in female embryos DNA methylation begins near 5.5 days and then rises, in both the embryo proper and the extraembryonic tissues, reaching nearly the adult level by 6.5 days.X-chromosome inactivation occurs in the inner cell mass of female mouse embryos between 5.5 and 6.5 days, at about the time of implantation (3, 10), a stage when the embryo consists of fewer than a thousand cells and the sex is not easily distinguishable. For this reason, only one prior study has been done correlating methylation with X-chromosome inactivation in the early embryo. Lock et al. (9) pooled many nonsexed embryos and found that methylation at sites in the first intron of the Hprt gene takes place between 9.5 and 13.5 days, well after the time of X-chromosome inactivation and the methylation we observe in the Pgk-1 gene.The promoter and first exon of Pgk-J are part of a CpG island that has been well characterized in both mice and humans (Fig. 1) (15) (Fig. 2). To assay for methylation, we made use of the fact that PCR amplification occurs only if the DNA between the two primer sites is intact. We chose PCR primers that flank H-7 and used a quantitative PCR procedure (20) to assay for HpaII-resistant (methylated) molecules. We had previously found, using this assay, that while male-embryo DNA was not amplified by PCR after HpaII digestion, female-embryo DNA was amplified, giving a signal 50% that of undigested DNA. Our interpretation was that site H-7 is methylated only on the inactive X chromosome (22). Figure 3 shows results of the HpaII-PCR assay on DNA from ectoplacental cone, extraembryonic ectoderm, and embryonic ectoderm dissected from two 6.5-day embryos, one male and one female. HpaII-resistant genomic molecules were present (giving band G in lanes marked +) in all three tissues of the female but not the male embryo. The larger fragments seen in the lanes without HpaII treatment came from M13 DNA, which was added as a carrier and also as a control for completeness of HpaII digestion. Band I in Fig. 3 is the PCR product of an internal standard, amplified by the same primers used for genomic DNA. The internal standard was a DNA fragment identical to genomic DNA except for a deletion internal to the primer binding sit...
To further our understanding of initiation and imprinting of X-chromosome inactivation, we have examined methylation of specific CpG sites of X-linked Pgk-1 and G6pd genes throughout female mouse development. Methylation occurs around the time of inactivation and earlier for Pgk-1, which is closer to the X-inactivation centre. In female primordial germ cells, the inactive X chromosome escapes methylation; this may underly the reversibility of inactivation at meiosis. Similarly, the genes are unmethylated on the inactive X chromosome in sperm; hence, the imprint specifying preferential X-inactivation in extra-embryonic tissues must reside elsewhere.
A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.
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