spectroscopic-super-resolution-imaging-dna-molecules-using-intrinsic

Spectroscopic Super-resolution Imaging of DNA Molecules using Intrinsic Contrast

Super-resolution imaging has revolutionized biological research by revealing structural details at the nanoscale. Most optical super-resolution methods ...

a-multilabel-single-molecule-localization-microscopy-protocol-for

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Super-resolution microscopy has dramatically advanced our ability to interrogate biological structures beyond the diffraction limit, making it ...

Northwestern University

Spectroscopic single-molecule localization microscopy (sSMLM) takes advantage of nanoscopy and spectroscopy, enabling sub-10 nm resolution as well as simultaneous multicolor imaging of multi-labeled samples. Reconstruction of raw sSMLM data using deep learning is a promising approach for visualizing the subcellular structures at the nanoscale.

Deep learning-based spectroscopic single-molecule localization microscopy.

In single cells, variably sized nanoscale chromatin structures are observed, but it is unknown whether these form a cohesive framework that regulates RNA transcription. Here, we demonstrate that the human genome is an emergent, self-assembling, reinforcement learning system. Conformationally defined heterogeneous, nanoscopic packing domains form by the interplay of transcription, nucleosome remodeling, and loop extrusion. We show that packing domains are not topologically associated domains. Instead, packing domains exist across a structure-function life cycle that couples heterochromatin and transcription in situ, explaining how heterochromatin enzyme inhibition can produce a paradoxical decrease in transcription by destabilizing domain cores. Applied to development and aging, we show the pairing of heterochromatin and transcription at myogenic genes that could be disrupted by nuclear swelling. In sum, packing domains represent a foundation to explore the interactions of chromatin and transcription at the single-cell level in human health.

Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system.

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. It is well established that cohesin is essential for TAD and loop connectivity features in Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with multiomic analysis and single-molecule localization microscopy, we study the role of cohesin in regulating the conformationally defined chromatin nanoscopic packing domains. Our results indicate that packing domains are not physical manifestation of TADs. Using electron microscopy, we found that only 20% of packing domains are lost upon RAD21 depletion. The effect of RAD21 depletion is restricted to small, poorly packed (nascent) packing domains. In addition, we present evidence that cohesin-mediated loop extrusion generates nascent domains that undergo maturation through nucleosome posttranslational modifications. Our results demonstrate that a 3D genomic structure, composed of packing domains, is generated through cohesin activity and nucleosome modifications.

Mature chromatin packing domains persist after RAD21 depletion in 3D.

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements and DNA damage. Nuclear blebs (i.e. herniations of the nuclear envelope) can be induced by (1) nuclear compression, (2) nuclear migration (e.g. cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated whether (1) packing domain organization is altered within nuclear blebs and (2) whether alteration in packing domain structure contributed to bleb formation. Using dual-partial wave spectroscopic microscopy, we show that chromatin-packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to what is seen in the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that chromatin within blebs is associated with a fragmented higher-order chromatin structure.

Nuclear blebs are associated with destabilized chromatin-packing domains.

Chromatin organization regulates transcription to influence cellular plasticity and cell fate. We explored whether chromatin nanoscale packing domains are involved in stemness and response to chemotherapy. Using an optical spectroscopic nanosensing technology we show that ovarian cancer-derived cancer stem cells (CSCs) display upregulation of nanoscale chromatin packing domains compared to non-CSCs. Cleavage under targets and tagmentation (CUT&Tag) sequencing with antibodies for repressive H3K27me3 and active H3K4me3 and H3K27ac marks mapped chromatin regions associated with differentially expressed genes. More poised genes marked by both H3K4me3 and H3K27me3 were identified in CSCs vs. non-CSCs, supporting increased transcriptional plasticity of CSCs. Pathways related to Wnt signaling and cytokine-cytokine receptor interaction were repressed in non-CSCs, while retinol metabolism and antioxidant response were activated in CSCs. Comparative transcriptomic analyses showed higher intercellular transcriptional heterogeneity at baseline in CSCs. In response to cisplatin, genes with low baseline expression levels underwent the highest upregulation in CSCs, demonstrating transcriptional plasticity under stress. Epigenome targeting drugs downregulated chromatin packing domains and promoted cellular differentiation. A disruptor of telomeric silencing 1-like (Dot1L) inhibitor blocked transcriptional plasticity, reversing stemness. These findings support that CSCs harbor upregulated chromatin packing domains, contributing to transcriptional and cell plasticity that epigenome modifiers can target.

Chromatin Organization Governs Transcriptional Response and Plasticity of Cancer Stem Cells.

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

Three-color single-molecule localization microscopy in chromatin.

Human tissues require a mechanism to generate durable, yet modifiable, transcriptional memories to sustain cell function across a lifetime. Previously, we demonstrated that nanoscale packing domains couple heterochromatin (cores) and euchromatin (outer zone) into unified reaction volumes that can generate transcriptional memory. In prior work, this framework demonstrated that RNA synthesis occurred within the ideal zone (intermediate density) portions of the domain. Naturally, this creates a question of where genes are positioned in relation to the packing domain architecture and which genetic material fills the domain core to sustain transcription. Here we propose that this could be solved by the encoded positioning of introns, intergenic segments, and exons as a projection of the functional packing layers of domains. This suggests that introns and intergenic segments are coupled to adjacent exons to generate coherent packing domain volumes. We illustrate how this organization would reconcile contradictions in epigenetic patterns, non-randomness in oncogenic mutations, and produce durable transcriptional memory. We conclude by showing that this genome geometry might have coincided with the rapid evolution of body-plan complexity, suggesting that chromatin geometry could be fundamental to metazoan evolution.

Geometrically encoded positioning of introns, intergenic segments, and exons in the human genome.

Cancer cells exhibit a remarkable resilience to cytotoxic stress, often adapting through transcriptional changes linked to alterations in chromatin structure. In several types of cancer, these adaptations involve epigenetic modifications and restructuring of topologically associating domains. However, the underlying principles by which chromatin architecture facilitates such adaptability across different cancers remain poorly understood. To investigate the role of chromatin in this process, we developed a physics-based model that connects chromatin organization to cell fate decisions, such as survival following chemotherapy. Our model builds on the observation that chromatin forms packing domains, which influence transcriptional activity through macromolecular crowding. The model accurately predicts chemoevasion in vitro, suggesting that changes in packing domains affect the likelihood of survival. Consistent results across diverse cancer types indicate that the model captures fundamental principles of chromatin-mediated adaptation, independent of the specific cancer or chemotherapy mechanisms involved. Based on these insights, we hypothesized that compounds capable of modulating packing domains, termed Transcriptional Plasticity Regulators (TPRs), could prevent cellular adaptation to chemotherapy. We conducted a proof-of-concept compound screen using live-cell chromatin imaging to identify several TPRs that synergistically enhanced chemotherapy-induced cell death. The most effective TPR significantly improved therapeutic outcomes in a patient-derived xenograft model of ovarian cancer. These findings underscore the central role of chromatin in cellular adaptation to cytotoxic stress and present a framework for enhancing cancer therapies, with broad potential across multiple cancer types.

Leveraging chromatin packing domains to target chemoevasion in vivo.

Human tissues require a mechanism to generate durable, yet modifiable, transcriptional memories to sustain cell function across a lifetime. Previously, it was demonstrated that nanoscale packing domains couple heterochromatin (cores) and euchromatin (outer zone) into unified reaction volumes that can generate transcriptional memory. In prior work, this framework demonstrates that RNA synthesis occurred within the ideal zone (intermediate density) portions of the domain. Naturally, this creates a question of where genes are positioned in relation to the packing domain architecture and which genetic material fills the domain core to sustain transcription. Here, it is proposed that this can be solved by the encoded positioning of introns, intergenic segments, and exons as a projection of the functional packing layers of domains. This suggests that introns and intergenic segments are coupled to adjacent exons to generate coherent packing domain volumes. How this organization will reconcile contradictions in epigenetic patterns, non-randomness in oncogenic mutations, and produce durable transcriptional memory is illustrated. The study concludes by showing that this genome geometry may have coincided with the rapid evolution of body-plan complexity, suggesting that chromatin geometry could be fundamental to metazoan evolution.

ArticleTotal : 2	Year
Publication title	2026
Publication title	2026

Article	Year
Deep learning-based spectroscopic single-molecule localization microscopy. Journal of biomedical optics\| PubMed ID: 38799979	2024
Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system. Science advances\| PubMed ID: 39792661	2025
Mature chromatin packing domains persist after RAD21 depletion in 3D. Science advances\| PubMed ID: 39854464	2025
Nuclear blebs are associated with destabilized chromatin-packing domains. Journal of cell science\| PubMed ID: 39878045	2025
Chromatin Organization Governs Transcriptional Response and Plasticity of Cancer Stem Cells. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 40051293	2025
Three-color single-molecule localization microscopy in chromatin. Light, science & applications\| PubMed ID: 40091134	2025
Geometrically encoded positioning of introns, intergenic segments, and exons in the human genome. bioRxiv : the preprint server for biology\| PubMed ID: 40501616	2025
Leveraging chromatin packing domains to target chemoevasion in vivo. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 40694328	2025
Geometrically Encoded Positioning of Introns, Intergenic Segments, and Exons in the Human Genome. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 41144753	2026

Ruyi Gong

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