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The way in which our DNA is folded into the cell nucleus can affect gene expression and determine cellular identity, but it can also regulate recombination and genome instability. Indeed, the disruption of key elements underlying the architecture of the nucleus, such as Topologically-Associated Domain (TAD) boundaries, has been shown to be the cause of genetically inherited diseases in some instances. How nuclear organization is first established after fertilization in mammals is largely unknown. In particular, the lack of approaches to observe in real time the different layers of nuclear organization has been a barrier for the full understanding of the dynamics and the impact of nuclear organization in vivo. Most importantly, the chromatin and structural pathways that regulate the initial folding of our genome into the nucleus as well as the impact of the resulting genome organization for key developmental and transcriptional processes in vivo, is largely unknown. We have recently shown that nuclear organization, particularly the association of specific genomic regions to the nuclear lamina, is established de novo in the newly formed embryo immediately after fertilization. Lamina- Associated Domains (LADs) precede the consolidation of TADs and compartments, suggesting that the association with the nuclear lamina may be the first nuclear architectural scaffold and set the framework for nuclear architecture. Here, to fulfill the outstanding need for methodologies to visualize specific chromosome domains, we will establish \u201cLivePaint\u201d, a technology to visualize the fundamental pillars of genome organization and their dynamics in real time (Aim 1). We propose to image the process of establishment of nuclear architecture by applying this tool to the mouse pre-implantation embryo, a full organism amenable to imaging and in which real time dynamics can be studied in their natural context. In parallel, we will deploy low input genomic approaches, such as DamID to identify the regulators of LAD establishment (Aim 2). Finally, using this information, we will perform perturbations to establish the role of LADs in regulating the establishment of nuclear architecture through both imaging and genomics approaches (Aim 3). We will also identify the impact of the disruption to the establishment of nuclear organization in vivo on epigenetic features, such as replication timing and gene expression and ultimately their impact on developmental competence. Through the perturbation and live imaging of the main determinants of nuclear organization in vivo, this proposal will establish the functional dependencies between the different layers of nuclear organization and will reveal their role in cellular function, gene expression and developmental progression at the beginning of mammalian development. The knowledge generated through these Aims will address outstanding challenges for our understanding of how the mammalian genome folds within the nuclear space in a physiological context and will reveal molecular regulators of this process.", "@id": "/awards/1U01DK127391-01/", "status": "current", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "status": "released", "aliases": ["torres-padilla-lab:lamnb1-damidseq-2cellembryo-ehmt2"], "accession": "4DNESF9768AS", "condition": "Ehmt2 transcript", "description": "LaminB1 DamID-seq on mouse 2-cell embryo with Ehmt2 transcript overexpressed", "date_created": "2024-06-11T17:49:06.805909+00:00", "submitted_by": {"error": "no view permissions"}, "dataset_label": "LaminB1 DamID-seq on mouse 2-cell embryos with transcripts overexpressed", "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2025-05-29T20:46:44.498292+00:00"}, "public_release": 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Notably, some perturbations exerted differential effects on  zygotes versus 2-cell-stage embryos. Moreover, embryos with disrupted LADs can  rebuild nuclear architecture at the 2-cell stage, indicating that the initial  establishment of LADs in zygotes might be dispensable for early development. Our  findings provide valuable insights into the functional interplay between  chromatin and structural components of the nucleus that guide genome-lamina  interactions during the earliest developmental stages.", "status": "current", "display_title": "Pal M et al. 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We find that perturbations  impacting histone H3 modifications, heterochromatin, and histone content are  crucial for the establishment of nuclear architecture in zygotes and/or  2-cell-stage embryos. Notably, some perturbations exerted differential effects on  zygotes versus 2-cell-stage embryos. Moreover, embryos with disrupted LADs can  rebuild nuclear architecture at the 2-cell stage, indicating that the initial  establishment of LADs in zygotes might be dispensable for early development. Our  findings provide valuable insights into the functional interplay between  chromatin and structural components of the nucleus that guide genome-lamina  interactions during the earliest developmental stages.", "ID": "PMID:40273908", "display_title": "Pal M et al. 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