Oral Presentation 47th Lorne Genome Conference 2026

Dynamics of embryonic DNA methylation in an Australian marsupial (133341)

Allegra Angeloni 1 2 , Jillian M Hammond 1 , Timothy J Peters 1 2 , Andre L. M. Reis 1 2 , Leah Kemp 1 , Timothy Amos 1 , Hasindu Gamaarachchi 1 3 , Sam Humphries 4 5 , Lynda Wilmott 6 , Suranjana Pal 6 , V. Pragathi Masamsetti 7 8 , Megan Weatherstone 9 , Kenny Chi Kin Ip 10 , Karina Pazaky 1 , Alice Steel 1 , Elly Walters 11 12 , Ning Liu 11 12 , Patrick Tam 7 8 , Jose M Polo 11 12 13 , Paul Waters 14 , Susan J Clark 1 2 , Linda Richards 6 , Andrew D Smith 15 , Heather Lee 4 5 , Ira W Deveson 1 2 , Oliver W Griffith 16 17 , Ksenia Skvortsova 1 14
  1. Garvan Institute of Medical Research, Sydney, NSW, Australia
  2. St Vincent's Clinical School, UNSW, Sydney, Australia
  3. School of Computer Science and Engineering, UNSW, Sydney, NSW, Australia
  4. Hunter Medical Research Institute, Newcastle, NSW, Australia
  5. School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia
  6. Department of Neuroscience, Washington University in St Louis School of Medicine, St Louis, Missouri, USA
  7. Embryology Research Unit, Children’s Medical Research Institute, Westmead, NSW, Australia
  8. School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
  9. Centre for Heart Research, The Westmead Institute for Medical Research, Sydney, NSW, Australia
  10. Children’s Cancer Institute, Sydney, NSW, Australia
  11. Adelaide Centre for Epigenetics, School of Biomedicine, The University of Adelaide, Adelaide, SA, Australia
  12. South Australian immunoGENomics Cancer Institute, The University of Adelaide, Adelaide, SA, Australia
  13. Department of Anatomy & Developmental Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, Australia
  14. School of Biotechnology and Biomolecular Sciences, UNSW, Sydney, NSW, Australia
  15. University of Southern California, Los Angeles, California, USA
  16. School of Natural Sciences, Macquarie University, Sydney, NSW, Australia
  17. Multi-omics Research Centre, Macquarie University, Sydney, NSW, Australia

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. In non-mammalian vertebrates like zebrafish, maternal 5mC profiles are modestly reprogrammed while high methylation is maintained throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. Although embryonic methylation is rapidly restored upon implantation, the trophectoderm, which forms the placenta, displays sustained hypomethylation, suggesting that this erasure is functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylation dynamics in marsupials, a therian lineage with a short-lived placenta.

The Australian fat-tailed dunnart (Sminthopsis crassicaudata) has recently emerged as a model marsupial species for comparative developmental biology, owing to their relative ease of husbandry, well-characterised embryogenesis, and unique placental biology. We assembled a near-complete, telomere-to-telomere (T2T) genome of the fat-tailed dunnart using long-read Nanopore sequencing and Hi-C. With this improved reference, we employed enzymatic methyl-seq and single-cell bisulfite sequencing to generate detailed epigenomic maps of dunnart embryonic development. We found the dunnart exhibits genome wide DNA demethylation at the blastocyst stage, but these changes occur in the trophectoderm only, suggesting that 5mC erasure in the placenta is an ancestral state in therian mammals. Furthermore, the T2T-level dunnart genome assembly enabled identification of sex chromosomes, uncovering extensive hypomethylation of the paternally-inherited inactive X chromosome in females and revealing the previously unannotated master regulator of X chromosome inactivation, lncRNA Rsx.

Our data indicate that while the use of genome-wide 5mC erasure differs between eutherian and marsupial lineages, 5mC erasure in extraembryonic tissue is ancestral to therian mammals and may be necessary to support placental development.