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.

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.

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.

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.

Detection of extracellular matrix modification in cancer models with inverse spectroscopic optical coherence tomography.

In cancer biology, there has been a recent effort to understand tumor formation in the context of the tissue microenvironment. In particular, recent progress has explored the mechanisms behind how changes in the cell-extracellular matrix ensemble influence progression of the disease. The extensive use of in vitro tissue culture models in simulant matrix has proven effective at studying such interactions, but modalities for non-invasively quantifying aspects of these systems are scant. We present the novel application of an imaging technique, Inverse Spectroscopic Optical Coherence Tomography, for the non-destructive measurement of in vitro biological samples during matrix remodeling. Our findings indicate that the nanoscale-sensitive mass density correlation shape factor D of cancer cells increases in response to a more crosslinked matrix. We present a facile technique for the non-invasive, quantitative study of the micro- and nano-scale structure of the extracellular matrix and its host cells.

The Greater Genomic Landscape: The Heterogeneous Evolution of Cancer.

Results have historically shown a broad plasticity in the origin of tumors and their functions, with significant heterogeneity observed in both morphologies and functional capabilities. Largely unknown, however, are the mechanisms by which these variations occur and how these events influence tumor formation and behavior. Contemporary views on the origin of tumors focus mainly on the role of particular sets of driver transformations, mutational or epigenetic, with the occurrence of the observed heterogeneity as an accidental byproduct of oncogenesis. As such, we present a hypothesis that tumors form due to heterogeneous adaptive selection in response to environmental stress through intrinsic genomic sampling mechanisms. Specifically, we propose that eukaryotic cells intrinsically explore their available genomic information, the greater genomic landscape (GGL), in response to stress under normal conditions, long before the formation of a cancerous lesion. Finally, considering the influence of chromatin heterogeneity on the GGL, we propose a new class of compounds, chromatin-protective therapies (CPT), which target the physical variations in chromatin topology. In this approach, CPTs reduce the overall information space available to limit the formation of tumors or the development of drug-resistant phenotypes. Cancer Res; 76(19); 5605-9. ©2016 AACR.