X chromosome inactivation is an epigenetic phenomenon in therian (eutherian and marsupial) mammals that results in the transcriptional silencing of one X chromosome in the somatic cells of females. In eutherian mammals, hypermethylation at gene promoters is a late, stabilizing step that maintains transcriptional silence on the inactive X. Previous single loci in different marsupial models demonstrated that there was no differential DNA methylation of promoters between the sexes, leading to the long standing conclusion that it plays no role in marsupial X-inactivation.
Using reduced representation bisulfite sequencing, we analysed CpG methylation to generate male and female methylation profiles in a marsupial representative (grey short-tailed opossum). In contrast to mouse, opossum displayed no differential DNA methylation between the sexes at promoters of genes subject to X-inactivation. However, regions adjacent to promoters of these genes were hypomethylated in females, “flattening” the DNA methylation profile. This is the first observation of non-random hypomethylation on the X in a female marsupial representative, which we propose acts as a silencing signal during X inactivation.