The cell nucleus is a highly dynamic and complex organelle that orchestrates fundamental cellular processes through its spatial organization. Far from being merely the repository of genetic information, it acts as a regulatory hub whose architecture profoundly influences transcription, RNA maturation and genome maintenance. Dissecting such a multilayered organization requires approaches that integrate molecular profiling with spatially resolved technologies capable of capturing nuclear architecture in situ. In this Review, we discuss classical and emerging imaging strategies that are transforming our understanding of nuclear organization across scales, from multiplexed and super-resolution light microscopy to barcoding-based spatial methods, live-cell imaging, and ultrastructural electron microscopy. Together, these methods are providing crucial insights into the localization and dynamics of RNAs and genomic regions within distinct compartments revealing how nuclear architecture governs genome function.

Mammalian chromatin architecture appears hierarchically organised and is composed of chromosome territories, compartments, topologically associating domains (TADs), loops and nucleosomes. The study of chromatin architecture has recently become prevalent due to the advancement of chromosome conformation capture (3C) and microscopy-based techniques. Many studies have predicted that TADs display important functional characteristics such as forming tissue and species-invariant regulatory domains, promoting co-regulation and insulating genes from aberrant regulation. Even with the increasing availability of data, the relationship between chromatin structures such as TADs, gene expression and function is still not fully understood. In this thesis, I use publicly available Hi-C data to rigorously assess this relationship. To further investigate the characteristics of TADs I compiled a re-annotated TAD dataset consisting of 13 human and 20 mouse genomes, covering 8 broad tissue/cell type groups. Using this TAD dataset as a foundation, I investigated the functional features of TADs in three main areas: tissue and species specificity, functional annotations, and phenotypic characteristics. Comparative analyses between TADs and randomly placed TADs revealed that there is some evidence that TADs are both tissue- and species-specific, genes which singly occupy a TAD are highly constrained and enriched for developmental functions, and genes sharing a CTCF TAD are not more functionally simi

The mammalian genome is hierarchically packaged into distinct functional units, including chromatin loops, topologically associating domains, compartments and chromosome territories. This structural organization is fundamentally important because it orchestrates essential nuclear functions that underpin normal cellular identity and organismal development. In this review, we synthesize current understanding of the intricate relationship between genome architecture and its critical biological roles. We discuss how hierarchical structures are dynamically established and maintained by architectural proteins, transcription factors, epigenetic regulators and non-coding RNAs via distinct mechanisms. Importantly, we focus on the functional consequences of three-dimensional (3D) genome organization and discuss how it modulates fundamental biological processes such as transcription, gene co-expression, epigenetic modification, DNA replication and repair. We also examine the dynamics of 3D genome organization during cellular differentiation, early embryonic development and organogenesis, followed by discussing how structural disruptions are mechanistically linked to various human diseases. Understanding the biological function of 3D genome organization is thus not only essential for deciphering fundamental nuclear processes but also holds significant promise for elucidating disease etiologies and developing effective therapeutics.

The field of 3D chromatin organization has advanced significantly in recent years, with a marked increase in research focusing on dynamics and viscoelastic properties of chromatin. The hierarchical model of chromatin organization, which spans from nucleosomes to loops, domains, compartments, and territories, has long been the standard framework for understanding chromatin structure. While this model successfully explains many aspects of chromatin organization, it falls short in explaining the dynamic aspects of chromatin. To capture the complexity of chromatin behavior, it is crucial to have a model that integrates both its structural organization and its dynamic properties. Similar to cell membranes, chromatin organization appears to be both mosaic and fluid, characterized by multiscale organization, conformational heterogeneity, and dynamic behavior. Adopting the fluid mosaic model for chromatin would offer a more comprehensive understanding of its functions, accommodating its complexity and dynamic nature.

Human pluripotent stem cells (hPSCs) are a promising source for regenerative medicine due to their self-renewal and differentiation capacities. However, genetic instability acquired during reprogramming and in vitro culture presents major safety challenges for clinical translation. Recurrent mutations, especially structural variants (SVs), are of particular concern as they can impair differentiation and increase tumorigenic risk. In this review, we establish and systematically explore a central causal axis: SVs-three dimensional (3D) genome disruption-safety of hPSC-based therapy. We propose that SVs critically compromise therapeutic safety by perturbing the 3D architecture of the genome, leading to pathogenic rewiring of enhancer-promoter interactions. This rewiring, exemplified by "enhancer hijacking" and "enhancer loss," can aberrantly activate oncogenes or silence tumor suppressors even in the absence of copy number variations. Thus, 3D genome disruption provides a key mechanistic explanation for SV-driven tumorigenic potential and impaired differentiation fidelity in hPSCs. By highlighting this causal axis, our review not only advances the mechanistic understanding of SV-associated risks but also provides actionable insights for the development of more rigorous quality standards for hPSC-based cell therapy products.