Expansion in situ genome sequencing links nuclear abnormalities to aberrant chromatin regulation.

Microscopy and genomics are used to characterize cell function, but approaches to connect the two types of information are lacking, particularly at subnuclear resolution. Here, we describe expansion in situ genome sequencing (ExIGS), a technology that enables sequencing of genomic DNA and super-resolution localization of nuclear proteins in single cells. Applying ExIGS to progeria-derived fibroblasts revealed that lamin abnormalities are linked to hotspots of aberrant chromatin regulation that may erode cell identity. Lamin was found to generally repress transcription, suggesting that variation in nuclear morphology may affect gene regulation across tissues and aged cells. These results demonstrate that ExIGS may serve as a generalizable platform with which to link nuclear abnormalities to gene regulation, offering insights into disease mechanisms. Editor's summary: The shape of the nucleus varies across cell types and can gradually develop visual abnormalities with aging or disease that can serve as diagnostic markers. Labade et al. developed a method to sequence the genome directly within physically expanded nuclei, enhancing imaging resolution. Using this approach, they examined how the three-dimensional organization of active and inactive chromatin changes in progeria, an accelerated aging disorder marked by nuclear lamina defects. This technique enables direct connections between nuclear morphology and alterations in genome architecture. —Di Jiang INTRODUCTION: Microscopy reveals the physical appearance of cells and enables clinical diagnoses, whereas genomics defines cell types using high-throughput molecular readouts. Although emerging spatial genomics approaches can link phenotypic disease markers to zenodo measurements, they often lack the resolution needed to precisely map associations within the cell nucleus, which is organized at nanometer scale. Thus, there remains a need for technologies capable of connecting high-resolution nuclear phenotypes with the spatial organization of the genome across cell types and diseases. RATIONALE: Multiplexed imaging methods can simultaneously localize genomic regions and nuclear proteins, but their accuracy in measuring DNA-protein interactions is constrained by the diffraction limit of optical microscopy and probe-targeting restrictions. To overcome these limitations, we integrated expansion microscopy (ExM) and in situ genome sequencing (IGS) into a single approach, enabling simultaneous nanoscale imaging of nuclear proteins and spatially resolved genomic sequencing within physically expanded samples. This new technology, which we call expansion in situ genome sequencing (ExIGS), can link high-resolution nuclear phenotypes to spatial genome organization within individual cells RESULTS: We first validated ExIGS in human fibroblast cells, demonstrating that our protocol uniformly expands nuclei while enhancing measurement of nanoscale DNA-protein interactions. We then applied ExIGS to fibroblasts derived from an individual with Hutchinson-Gilford progeria syndrome, allowing us to study how the characteristic nuclear abnormalities of this disease impact spatial genome organization. On the imaging side, expansion immunofluorescence of lamin A/C enabled us to quantitatively characterize lamin defects, including invaginations extending into the nuclear interior, which progressively accumulate in progeria fibroblasts across successive passages. On the sequencing side, three-dimensional (3D) localization analysis of genomic DNA fragments revealed that these defects primarily disrupt the functional compartmentalization of chromatin at local hotspots within individual nuclei rather than causing global disruption. Finally, paired expansion imaging of lamin A/C and RNA polymerase II revealed that lamin structures are generally associated with transcriptional repression and exhibit structural variation not only in progeria but also across normal tissue and aging contexts. CONCLUSION: ExIGS unifies high-resolution imaging and sequencing within expanded nuclei, enabling direct characterization of how variations in nuclear morphology affect spatial genome organization. The local chromatin disruption hotspots that we observed in progeria fibroblasts provide new opportunities to investigate, such as whether lamin defects are also linked to chromatin dysregulation in normal aging. Moreover, our finding that lamin is broadly repressive regardless of structural configuration suggests that variation in nuclear shape may represent an underappreciated mechanism of gene regulation across biological contexts. We anticipate that ExIGS will serve as a generalizable imaging and sequencing platform for connecting high-resolution phenotypic variation to multiomic readouts across cells, tissues, and organisms. ExIGS links nuclear morphology to spatial genome organization at high resolution.: In ExIGS, nuclei are physically enlarged in an expandable hydrogel, nuclear proteins are imaged using immunofluorescence, and 3D genomic locations are identified using in situ sequencing. Applied to progeria, ExIGS enables high-resolution quantification of how characteristic lamin abnormalities affect 3D chromosome structures. [ABSTRACT FROM AUTHOR]