Mosaic methylation of Xist gene before chromosome inactivation in undifferentiated female mouse embryonic stem and embryonic germ cells.

Epigenetic modification is implicated in the choice of the X chromosome to be inactivated in the mouse. In order to gain more insight into the nature of such modification, we carried out a series of experiments using undifferentiated mouse cell lines as a model system. Not only the paternally derived X (XP) chromosome, but the maternally derived one (XM) was inactivated in the outer layer of the balloon-like cystic embryoid body probably corresponding to the yolk sac endoderm of the post-implantation embryo in which XP is preferentially inactivated. Hence, it is likely that the imprint responsible for the nonrandom XP inactivation in early mouse development has been erased or masked in female ES cells. CpG sites in the 5' region of the Xist gene were partially methylated in female ES and EG and parthenogenetic ES cell lines as in the female somatic cell in which the silent Xist allele on the active X is fully methylated, whereas the expressed allele on the inactive X is completely unmethylated. In the case of undifferentiated ES cells, however, methylation was not differential between two Xist alleles. This observation was supported by the demonstration that single-cell clones derived from female ES cell lines were not characterized by either allele specific Xist methylation or nonrandom X inactivation upon cell differentiation. Apparently these findings are at variance with the view that Xist expression and X inactivation are controlled by preemptive methylation in undifferentiated ES cells and probably in epiblast.