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

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is well characterized in eutherian mammals. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI 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. First, tissue-specific differential DNA methylation in marsupials primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. 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 XCI, 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 functionally conserved patterns of XCI in two deep branches of mammals.