Divergent DNA methylation signatures underlying X chromosome regulation in marsupials and eutherians

The phenomenon of X chromosome inactivation (XCI) mediated by sex-specific differences in DNA methylation is well characterized in eutherian mammals. Although XCI is shared between eutherians and marsupials, the role of marsupial DNA methylation in this process remains contested. Here we examine genome-wide signatures of DNA methylation from methylation maps across fives tissues from a male and female koala (Phascolarctos cinereus) and present the first whole genome, multi-tissue marsupial "methylome atlas." Using these novel data, we elucidate divergent versus common features of marsupial and eutherian DNA methylation and XCI. First, tissue-specific differential DNA methylation in marsupials primarily occurs in gene bodies. Second, the marsupial X chromosome is significantly globally less methylated (hypomethylated) in females compared to males. We show that this pattern is also observed in eutherian X chromosomes. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial X chromosome inactivation, is consistent with the epigenetic regulation of female- (and presumably inactive X chromosome-) specific expression. Finally, we utilize the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5 %) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to the functionally conserved pattern of X chromosome inactivation in two deep branches of mammals.