Methylation profiles of DXPas34 during the onset of X-inactivation

X chromosome inactivation is controlled by the cis-acting X-inactivation centre (Xic). In addition to initiating inactivation, Xic, which includes the Xist gene, is involved in both a counting process that Senses the number of X chromosomes and the choice of X chromosome to inactivate. Controlling elements lying 3' to Xist include the DXPas34 locus. Deletion of DXPas34 in undifferentiated embryonic stem (ES) cells eliminates expression of both Xist and the antisense transcript Tsix, thought to initiate from a CpG island lying close to, but telomeric to, the DXPas34 locus itself. Deletion of DXPas34 leads to nonrandom inactivation on ES cell differentiation and disrupts imprinted X-inactivation in vivo. In order to investigate the role of methylation at DXPas34 in the initial steps of X-inactivation, we studied its methylation status during pre- and post-implantation embryonic development and ES cell differentiation, using the bisulphite sequencing technique, Analysis of the methylation status of both the DXPas34 locus and the associated downstream CpG island shows that extensive hypermethylation of the DXPas34 locus is a relatively late event in differentiation and embryogenesis. We conclude that methylation of DXPas34 cannot be the X chromosome imprint, nor can it be involved in the parent-of-origin effects associated with deletion of the DXPas34 locus and the neighbouring CpG island.