In a groundbreaking study revealing the intricacies of mammalian embryogenesis, researchers have uncovered striking differences in DNA methylation dynamics driving X chromosome inactivation (XCI) between marsupials and eutherians. This discovery not only illuminates previously obscure epigenetic mechanisms but redefines our understanding of imprinting and dosage compensation in mammalian species, with potential far-reaching implications for genetics and developmental biology.
X chromosome inactivation is a vital process that balances gene expression in female mammals by silencing one of their two X chromosomes. While in most eutherians, such as humans and other placental mammals, this silencing occurs randomly in the early embryo, the mouse serves as a notable exception. In mice, the paternal X chromosome is selectively silenced through an imprinted mechanism during preimplantation, preceding the random XCI that happens later in embryonic development. However, the epigenetic underpinning of this imprinting contrasts sharply with mechanisms observed in other species.
Investigations have demonstrated that imprinted XCI in mice interestingly operates independently of DNA methylation. Instead, it relies on the repressive histone modification H3K27me3 to silence the maternal Xist allele. This epigenetic hallmark guides the selective inactivation process, showcasing a methylation-independent mode of imprinting that has challenged the classical understanding of DNA methylation as a universal imprinting regulator. The subtleties of this mechanism raise profound questions about how marsupials, which diverged early from the eutherian lineage, manage XCI imprinting at an epigenetic level.
In a meticulously designed study focusing on the opossum, a representative marsupial, scientists identified a Differentially Methylated Region (DMR) overlapping the RSX promoter that exhibits distinct methylation patterns in gametes. Specifically, this promoter region is highly methylated in oocytes but remains hypomethylated in sperm. Notably, this pattern persists into female embryos and adult tissues, implying a heritable epigenetic signature that potentially instructs imprinted XCI in marsupials. This finding contrasts with murine models and suggests a DNA methylation-dependent imprinting mechanism in marsupials.
To probe the functional consequences of DNA methylation at the RSX promoter, researchers employed CRISPR-based genome editing to delete the DNA methyltransferases, DNMT1A and DNMT1B, collectively referred to as DNMT1, in male opossum fibroblasts. DNMT1 enzymes are essential for maintaining DNA methylation after replication, thus their ablation offers a powerful tool to dissect methylation-dependent gene regulation. This epigenetic editing led to a widespread reduction in DNA methylation, including hypomethylation at the RSX promoter, confirming the critical role of DNMT1 in sustaining methylation landscapes in these cells.
Remarkably, loss of DNMT1 triggered ectopic expression of the RSX gene, which is normally silent in male opossums due to the single X chromosome and lack of inactivation. Quantitative PCR analysis revealed robust activation of the RSX transcript, accompanied by the formation of distinct RSX RNA clouds within the nuclei of these fibroblasts, visualized through RNA fluorescence in situ hybridization. This reactivation highlights a cause-effect relationship between DNA methylation and repression of RSX, underlining the instructive role of methylation in maintaining normal expression patterns.
Ectopic RSX expression in male cells had further biological consequences, notably the suppression of X-linked gene activity. Transcriptomic analyses showed X chromosome genes were disproportionately downregulated following DNMT1 ablation. Concomitantly, the overall X-to-autosome expression ratio decreased, signaling a shift in dosage balance likely mediated by aberrant RSX function. These findings indicate that RSX RNA, akin to its eutherian counterpart Xist, orchestrates chromosomal silencing and that its epigenetic control via DNA methylation is pivotal to ensuring proper gene dosage.
Beyond the X chromosome, global DNA methylation loss also unleashed widespread transposable element activation, as seen by increased expression levels of multiple families of mobile genetic elements. This surge points to the central role DNA methylation plays in genome stability, repressing potentially deleterious elements that, if mobilized, could disrupt genomic integrity. Such insights emphasize the dual regulatory functions of methylation in epigenetic gene silencing and transposon control.
Further reinforcing the generality of DNA methylation in imprinting regulation, the study examined expression of the autosomal H19 locus, which is imprinted in marsupials but absent in the opossum genome assembly used in prior screens. Upon DNMT1 deletion, H19 expression levels significantly increased, suggesting that DNA methylation acts broadly across different imprinted loci beyond the X chromosome. This expansion of methylation’s role invites reconsideration of imprinted gene control and reveals evolutionary divergence in epigenetic strategies in marsupials versus placental mammals.
Collectively, these elegant experiments illustrate that, unlike the mouse model, marsupials leverage DNA methylation at the RSX promoter to imprint X chromosome inactivation. This methylation imprint is set during gametogenesis and maintained into embryonic development, dictating allele-specific expression patterns. The interplay between methylation marks and expression of long non-coding RNAs such as RSX presents an evolutionary distinct but functionally analogous system to the eutherian Xist-based paradigm.
These findings also have broader implications for understanding mammalian epigenetics and evolutionary biology. By delineating the divergent mechanisms of dosage compensation and imprinting, this research opens avenues for exploring how epigenetic pathways adapt across species. The use of cutting-edge CRISPR genome editing combined with epigenomic profiling sets a new standard for dissecting functional epigenetic regulation in non-traditional model organisms.
As we refine our grasp on the molecular choreography governing early development, the marsupial model provides a compelling window into alternative strategies evolution has crafted. Epigenetic landscapes are not ubiquitously conserved but reflect lineage-specific adaptations, reminding us that mammalian biology is richly diverse and complex. The revelation that DNA methylation orchestrates RSX silencing in marsupials versus H3K27me3-dependent Xist silencing in mice underscores the dynamic interplay of epigenetic modifications across taxa.
In summary, this study shines light on the crucial but divergent roles that DNA methylation plays in regulating X chromosome inactivation and imprinting across mammalian lineages. It challenges longstanding assumptions derived from murine models and enriches our understanding of epigenetic control mechanisms. As epigenetics continues to unravel the complexities of gene regulation, studies like this reaffirm the value of comparative approaches to decode the evolutionary tapestry of life.
Subject of Research: Epigenetic regulation of X chromosome inactivation and imprinting in marsupial and eutherian embryos
Article Title: Divergent DNA methylation dynamics in marsupial and eutherian embryos
Article References:
Leeke, B.J., Varsally, W., Ogushi, S. et al. Divergent DNA methylation dynamics in marsupial and eutherian embryos. Nature (2025). https://doi.org/10.1038/s41586-025-08992-2
Image Credits: AI Generated
