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. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.

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. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

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. Applying CPMC model predictions to transcriptional data from cancer patients, we identify an inverse relationship between patient survival and phenotypic plasticity of tumor 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. These functional modules need to be connected and isolated by an open backbone that results in porous and heterogeneous packing in a quasi-self-similar manner, as revealed by our electron and optical imaging. Our multiscale theoretical and experimental results suggest the existence of higher-order universal folding principles for a disordered chromatin fiber to avoid entanglement and fulfill its biological functions.

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. Therefore, to capture such dynamic crowding and investigate its influence on transcription, we employ a three-pronged, systems-molecular approach. A system of chemical reactions represents the transcription pathway, the rates of which are determined by molecular-scale simulations; Brownian dynamics and Monte Carlo simulations quantify protein diffusion and DNA-protein binding affinity, dependent on macromolecular density. Altogether, this approach shows that transcription depends critically on dynamic crowding as the gene expression resultant from dynamic crowding can be profoundly different than that of steady-state crowding. In fact, expression levels can display both amplification and suppression and are notably different for genes or gene populations with different chemical and structural properties. These properties can be exploited to impose circadian expression, which is asymmetric and varies in strength, or to explain expression in cells under biomechanical stress. Therefore, this work demonstrates that dynamic crowding nontrivially alters transcription kinetics and presents dynamic crowding within the bulk nuclear nanoenvironment as a novel regulatory framework for gene expression.

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. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through MSCs and consequent changes to histone modification state and chromatin-based nuclear rigidity.

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. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two live cell models: a drug-based model of Hela cells differentially treated with daunorubicin and a cell line comparison model of two cells lines with inherently distinct chromatin organizations. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology.

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. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through mechanosensitive ion channels and consequent changes to histone modification state and chromatin-based nuclear rigidity.

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. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells.

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. With a novel spectral regression algorithm that identifies the spectroscopic fingerprints of neighboring molecules that were previously indistinguishable, we demonstrate the utility of SICLON by visualizing unlabeled poly-nucleotides and linear single-stranded DNA fibers with a resolution of 6.2 nm.

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]). The low-risk history groups include patients with a history of NDA. The high-risk history groups include patients with either a history of AA or colorectal cancer (CRC). PWS is a nanoscale-sensitive imaging technique which measures the organization of intracellular structure. Previous studies have shown that PWS is sensitive to changes in the higher-order (20-200 nm) chromatin topology that occur due to field carcinogenesis within histologically normal cells. The results of this study show that these nanoscale structural alterations are correlated with a patient's colonic history, which suggests that PWS can detect altered field carcinogenic signatures even in patients with negative colonoscopies. Furthermore, we developed a model to calculate the 5-year risk of developing CRC for each patient group. We found that our data fit this model remarkably well (R2  = 0.946). This correlation suggests that PWS could potentially be used to monitor CRC progression less invasively and in patients without adenomas, which opens PWS to many potential cancer care applications.

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. Recent progress in partial wave spectroscopic (PWS) microscopy enables real-time measurement of higher-order chromatin organization within label-free live cells. Specifically, PWS quantifies the nanoscale variations in mass density (heterogeneity) within the cell. These advancements have made it possible to study the functional role of chromatin topology, such as its regulation of the global transcriptional state of the cell and its role in the development of cancer. In this chapter, the importance of studying chromatin topology is explained, the theory and instrumentation of PWS are described, the measurements and analysis processes for PWS are laid out in detail, and common issues, troubleshooting steps, and validation techniques are provided.

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. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson-Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity.

The transformation of the nuclear nanoarchitecture in human field carcinogenesis.

Morphological alterations of the nuclear texture are a hallmark of carcinogenesis. At later stages of disease, these changes are well characterized and detectable by light microscopy. Evidence suggests that similar albeit nanoscopic alterations develop at the predysplastic stages of carcinogenesis. Using the novel optical technique partial wave spectroscopic microscopy, we identified profound changes in the nanoscale chromatin topology in microscopically normal tissue as a common event in the field carcinogenesis of many cancers. In particular, higher-order chromatin structure at supranucleosomal length scales (20-200 nm) becomes exceedingly heterogeneous, a measure we quantify using the disorder strength (Ld ) of the spatial arrangement of chromatin density. Here, we review partial wave spectroscopic nanocytology clinical studies and the technology's promise as an early cancer screening technology.

The effects of chemical fixation on the cellular nanostructure.

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.

Stochastic fluorescence switching of nucleic acids under visible light illumination.

We report detailed characterizations of stochastic fluorescence switching of unmodified nucleic acids under visible light illumination. Although the fluorescent emission from nucleic acids under the visible light illumination has long been overlooked due to their apparent low absorption cross section, our quantitative characterizations reveal the high quantum yield and high photon count in individual fluorescence emission events of nucleic acids at physiological concentrations. Owing to these characteristics, the stochastic fluorescence switching of nucleic acids could be comparable to that of some of the most potent exogenous fluorescence probes for localization-based super-resolution imaging. Therefore, utilizing the principle of single-molecule photon-localization microscopy, native nucleic acids could be ideal candidates for optical label-free super-resolution imaging.

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. Because transcriptional interactions are essentially chemical reactions, they depend largely on the local physical nanoenvironment. We show that the regulation of the chromatin nanoenvironment allows for the predictable modulation of global patterns in gene expression. In particular, we show that the rational modulation of chromatin density fluctuations can lead to a decrease in global transcriptional activity and intercellular transcriptional heterogeneity in cancer cells during chemotherapeutic responses to achieve near-complete cancer cell killing in vitro. Our findings represent a 'macrogenomic engineering' approach to modulating the physical structure of chromatin for whole-scale transcriptional modulation.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy.

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.