Methylation profiles of DXPas34 during the onset of X-inactivation

X chromosome inactivation is controlled by the cis-acting X-inactivation ce ntre (Xic). In addition to initiating inactivation, Xic, which includes the Xist gene, is involved in both a counting process that Senses the number o f X chromosomes and the choice of X chromosome to inactivate. Controlling e lements lying 3' to Xist include the DXPas34 locus. Deletion of DXPas34 in undifferentiated embryonic stem (ES) cells eliminates expression of both Xi st and the antisense transcript Tsix, thought to initiate from a CpG island lying close to, but telomeric to, the DXPas34 locus itself. Deletion of DX Pas34 leads to nonrandom inactivation on ES cell differentiation and disrup ts imprinted X-inactivation in vivo. In order to investigate the role of me thylation 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, An alysis of the methylation status of both the DXPas34 locus and the associat ed downstream CpG island shows that extensive hypermethylation of the DXPas 34 locus is a relatively late event in differentiation and embryogenesis. W e conclude that methylation of DXPas34 cannot be the X chromosome imprint, nor can it be involved in the parent-of-origin effects associated with dele tion of the DXPas34 locus and the neighbouring CpG island.