Cytosine methylation is an epigenetic modification that plays a role in regulation of gene transcription. Methylation, particularly at promoter CpG-islands, can lead to stable silencing of the associated gene. In mammals, DNA methylation has several well characterized regulatory functions, including genomic imprinting and the chromosome-wide epigenetic silencing of the X chromosome (called X-chromosome inactivation; XCI). XCI is part of a dosage compensation system in therian (eutherian and marsupial) mammals that results in almost equal average transcriptional output from the X chromosome between the sexes in eutherian and marsupial mammals. In contrast, platypus (males have 5 Xs and 5 Ys: females 5 pairs of Xs) and chicken (with a ZW female: ZZ male sex chromosome system), dosage compensation appears less efficient, where average Z/X transcriptional output is higher in the homogametic sex than in the heterogametic sex. Nevertheless, we recently demonstrated that alleles on one X/Z is also silenced in the homogametic sex of these species.

DNA methylation is a late and stabilizing step in maintaining transcriptional silence of the X in eutherian mammals, but there are limited detailed data about DNA methylation in marsupials, monotreme and birds. Here we present the first genome wide approach to study DNA methylation in non-eutherian representatives from three amniote vertebrate lineages, each with independently evolved dosage compensation systems. We examine differential CpG methylation between males and females in distantly related taxa to identify the importance of methylation in amnoite vertebrate dosage compensation.