Molecular mechanisms underlying the regulation of nuclear organization in early mouse embryos

Pal, Mrinmoy (2025): Molecular mechanisms underlying the regulation of nuclear organization in early mouse embryos. Dissertation, LMU München: Fakultät für Biologie

Abstract

Mammalian gametogenesis and early development involve extensive epigenomic reorganization, offering a unique opportunity to address important yet underexplored questions central to reproductive biology and regenerative medicine. How does the maternal germline reprogram to produce a fertilization-competent egg? How is the epigenome established following fertilization? What molecular pathways orchestrate this process in vivo? What are the dependencies between the different layers of the epigenome? How do these dynamic changes functionally influence developmental plasticity and cell fate? To address these questions, the thesis employs mouse oocytes and preimplantation embryos as a model for early mammalian development. The low-input DamID technique was adapted to map genome interactions with the nuclear lamina across different developmental stages, focusing on lamina-associated domains (LADs). We found that autosomal LADs are already undetectable in growing oocytes. These gene desert regions, usually heterochromatic in other cell types, contain oocyte-specific enhancer elements that regulate folliculogenesis. After fertilization, LADs undergo gradual yet dynamic reorganization during the maternal-to-zygotic transition, both following the first mitosis and throughout the progression of the second cell cycle. This repositioning correlates with the expression of genes and transposable elements in 2-cell stage embryos. Inhibition of transcription during zygotic genome activation (ZGA) impairs the correct rearrangement of LADs, leading to atypical features of lamina-associated chromatin. Single-cell Repli-seq showed that DNA replication occurs according to a less-defined pattern in the zygote, and the replication timing (RT) program gradually consolidates with developmental progression. Our findings suggest that LAD formation precedes and potentially predisposes the partitioning of the genome into early and late replicating domains. We identified RIF1 as a key regulator of replication timing consolidation in vivo, with its depletion resulting in a less coordinated RT program in 4-cell stage embryos and beyond. Intriguingly, the changes in RT in RIF1-depleted embryos are uncoupled from changes in genome–lamina association. A targeted screen identified chromatin pathways required for de novo LAD establishment. Although LADs are not inherited from oocytes, this work suggests that the maternal germline carries epigenetic bookmarking to guide the establishment of nuclear organization in zygotes. Our observations indicate that the absence of a constitutive heterochromatin pathway permits the distinctive LAD fragmentation at the 2-cell stage to coexist with a non-canonical chromatin landscape. We propose that LAD boundaries are reorganized based on positional information from H3K4me3 and H3K9me3 domains, which counteract each other. Remarkably, the initial establishment of LADs in zygotes is not essential for preimplantation development, as embryos can reconstruct their nuclear architecture by the 2-cell stage. In summary, this thesis provides valuable insights into the molecular understanding of epigenome establishment and highlights hierarchies between embryonic chromatin, 3D nuclear organization, and genome function.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Development, Epigenetics, Nuclear architecture, Maternal-to-zygotic transition
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. Juli 2025
• 1. Bericht­erstatter:in: Torres-Padilla, Maria-Elena