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

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.

Nuclear blebs are associated with destabilized chromatin-packing domains.

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements and DNA damage. Nuclear blebs (i.e.

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

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.

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

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.

Leveraging chromatin packing domains to target chemoevasion .

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 (TADs). However, the underlying principles by which chromatin architecture facilitates such adaptability across different cancers remain poorly understood.

Local volume concentration, packing domains, and scaling properties of chromatin.

We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions. The SR-EV generate conformationally defined domains observed by single-cell imaging techniques. From nucleosome to chromosome scales, the model captures the overall chromatin organization as a corrugated system, with dense and dilute regions alternating in a manner that resembles the mixing of two disordered bi-continuous phases.

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure.

Nuclear blebs are associated with destabilized chromatin packing domains.

Unlabelled: Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e.

Depletion of lamins B1 and B2 promotes chromatin mobility and induces differential gene expression by a mesoscale-motion-dependent mechanism.

Background: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology.

Local Volume Concentration, Packing Domains and Scaling Properties of Chromatin.

We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions. The SR-EV generate conformationally-defined domains observed by single cell imaging techniques. From nucleosome to chromosome scales, the model captures the overall chromatin organization as a corrugated system, with dense and dilute regions alternating in a manner that resembles the mixing of two disordered bi-continuous phases.

Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function.

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells.

Physical and data structure of 3D genome.

With the textbook view of chromatin folding based on the 30-nm fiber being challenged, it has been proposed that interphase DNA has an irregular 10-nm nucleosome polymer structure whose folding philosophy is unknown. Nevertheless, experimental advances suggest that this irregular packing is associated with many nontrivial physical properties that are puzzling from a polymer physics point of view. Here, we show that the reconciliation of these exotic properties necessitates modularizing three-dimensional genome into tree data structures on top of, and in striking contrast to, the linear topology of DNA double helix.

Disordered chromatin packing regulates phenotypic plasticity.

Three-dimensional supranucleosomal chromatin packing plays a profound role in modulating gene expression by regulating transcription reactions through mechanisms such as gene accessibility, binding affinities, and molecular diffusion. Here, we use a computational model that integrates disordered chromatin packing (CP) with local macromolecular crowding (MC) to study how physical factors, including chromatin density, the scaling of chromatin packing, and the size of chromatin packing domains, influence gene expression. We computationally and experimentally identify a major role of these physical factors, specifically chromatin packing scaling, in regulating phenotypic plasticity, determining responsiveness to external stressors by influencing both intercellular transcriptional malleability and heterogeneity.

Dynamic Crowding Regulates Transcription.

The nuclear environment is highly crowded by biological macromolecules, including chromatin and mobile proteins, which alter the kinetics and efficiency of transcriptional machinery. These alterations have been described, both theoretically and experimentally, for steady-state crowding densities; however, temporal changes in crowding density ("dynamic crowding") have yet to be integrated with gene expression. Dynamic crowding is pertinent to nuclear biology because processes such as chromatin translocation and protein diffusion lend to highly mobile biological crowders.

Physicochemical mechanotransduction alters nuclear shape and mechanics via heterochromatin formation.

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels (MSCs), without large-scale cell stretching.

Preservation of cellular nano-architecture by the process of chemical fixation for nanopathology.

Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy and have not been examined with nanoscale sensitive imaging techniques.

Physicochemical mechanotransduction alters nuclear shape and mechanics via heterochromatin formation.

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels, without large-scale cell stretching.

Multimodal interference-based imaging of nanoscale structure and macromolecular motion uncovers UV induced cellular paroxysm.

Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching.

Spectral contrast optical coherence tomography angiography enables single-scan vessel imaging.

Optical coherence tomography angiography relies on motion for contrast and requires at least two data acquisitions per pointwise scanning location. We present a method termed spectral contrast optical coherence tomography angiography using visible light that relies on the spectral signatures of blood for angiography from a single scan using endogenous contrast. We demonstrate the molecular sensitivity of this method, which enables lymphatic vessel, blood, and tissue discrimination.

Sub-10-nm imaging of nucleic acids using spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON).

Elucidating chromatin structure in vitro requires resolution below 10 nm to visualize the mononucleosome has been an ongoing challenge. In this work, we achieve sub-10-nm imaging of nucleic acids via spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON) without the use of external labels. SICLON leverages two key innovations: using endogenous nucleotides as the emission source and a custom-made imaging system that can simultaneously record the position and optical spectra of emitting molecules.

Correlating colorectal cancer risk with field carcinogenesis progression using partial wave spectroscopic microscopy.

Prior to the development of a localized cancerous tumor, diffuse molecular, and structural alterations occur throughout an organ due to genetic, environmental, and lifestyle factors. This process is known as field carcinogenesis. In this study, we used partial wave spectroscopic (PWS) microscopy to explore the progression of field carcinogenesis by measuring samples collected from 190 patients with a range of colonic history (no history, low-risk history, and high-risk history) and current colon health (healthy, nondiminutive adenomas (NDA; ≥5 mm and <10 mm), and advanced adenoma [AA; ≥10 mm, HGD, or >25% villous features]).

Measuring Nanoscale Chromatin Heterogeneity with Partial Wave Spectroscopic Microscopy.

Despite extensive research in the area, current understanding of the structural organization of higher-order chromatin topology (between 20 and 200 nm) is limited due to a lack of proper imaging techniques at these length scales. The organization of chromatin at these scales defines the physical context (nanoenvironment) in which many important biological processes occur. Improving our understanding of the nanoenvironment is crucial because it has been shown to play a critical functional role in the regulation of chemical reactions.

Macrogenomic engineering via modulation of the scaling of chromatin packing density.

Many human diseases result from the dysregulation of the complex interactions between tens to thousands of genes. However, approaches for the transcriptional modulation of many genes simultaneously in a predictive manner are lacking. Here, through the combination of simulations, systems modelling and in vitro experiments, we provide a physical regulatory framework based on chromatin packing-density heterogeneity for modulating the genomic information space.

Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins.

Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed "blebs" are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations.