The timing and pattern of methylation of the CpG island in the X-chromosome gene, Hprt, was examined using bisulfite methods to assess the occurrence of DNA cytosine methylation at the onset of X-chromosome inactivation (XCI). The Hprt sequences were extensively methylated in 4.5 dpc blastocysts, and the levels of methylation progressively decreased to 7.5 dpc. Adult patterns of methylation were established in the embryonic tissues after 7.5 dpc. By contrast, methylation continued to decrease in extraembryonic lineages and at 13.5 days was not detectable on the paternal Hprt sequences in the yolk sac endoderm. In the bisulfite-treated coding strand, numerous G to A transitions and CpTpG methylations were observed that were unique to the methylated or nonmethylated Hprt sequences, respectively, in early development.
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