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The authors examine Rome and China in the third century BCE, empires that sustained state power for centuries. They delve into the militant monotheism of Byzantium, the Islamic Caliphates, and the short-lived Carolingians.

Chromatin organization over multiple length scales plays a critical role in the regulation of transcription. Deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro . Herein, we introduce ChromSTEM, a method that utilizes high-angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM for an in-depth quantification of 3D chromatin conformation with high spatial resolution and contrast, allowing for characterization of higher-order chromatin structure almost down to the level of the DNA base pair. Employing mass scaling analysis on ChromSTEM mass density tomograms, we observed that chromatin forms spatially well-defined higher-order domains, around 80 nm in radius. Within domains, chromatin exhibits a polymeric fractal-like behavior and a radially decreasing mass-density from the center to the periphery. Unlike other nanoimaging and analysis techniques, we demonstrate that our unique combination of this high-resolution imaging technique with polymer physics-based analysis enables us to (i) investigate the chromatin conformation within packing domains and (ii) quantify statistical descriptors of chromatin structure that are relevant to transcription. We observe that packing domains have heterogeneous morphological properties even within the same cell line, underlying the potential role of statistical chromatin packing in regulating gene expression within eukaryotic nuclei.

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. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

Topographical cues on cells can, through contact guidance, alter cellular plasticity and accelerate the regeneration of cultured tissue. Here we show how changes in the nuclear and cellular morphologies of human mesenchymal stromal cells induced by micropillar patterns via contact guidance influence the conformation of the cells’ chromatin and their osteogenic differentiation in vitro and in vivo. The micropillars impacted nuclear architecture, lamin A/C multimerization and 3D chromatin conformation, and the ensuing transcriptional reprogramming enhanced the cells’ responsiveness to osteogenic differentiation factors and decreased their plasticity and off-target differentiation. In mice with critical-size cranial defects, implants with micropillar patterns inducing nuclear constriction altered the cells’ chromatin conformation and enhanced bone regeneration without the need for exogenous signalling molecules. Our findings suggest that medical device topographies could be designed to facilitate bone regeneration via chromatin reprogramming. Micropillar patterns causing changes in the nuclear and cellular morphologies of human mesenchymal stromal cells influence the conformation of the cells’ chromatin and their osteogenic differentiation in vitro and in mice.

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. We present a protocol enabling multi-color chromatin SMLM, revealing transcription and enhancer-associated euchromatin organizes around heterochromatic cores into functional volumes. This significantly advances nanoscopic molecular imaging within the nucleus.

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. Through a combination of transcriptomic and histone mapping studies integrated with chromatin imaging, we define how chromatin organization in nanoscale packing domains fine tunes the plasticity of cancer stem cells (CSCs) driving evasion from and survival after chemotherapy. Our results link the physical conformation of chromatin to the transcriptional program of CSCs enabling stemness features and evasion from chemotherapy.

Background Since oocyte donors are typically young and believed to be a source of highly competent gametes, donor oocyte IVF is considered to be an effective treatment for diminished ovarian reserve. However, the aneuploidy rate for embryos originating from anonymously donated oocytes remains incompletely characterized. Here, comprehensive chromosomal screening results were reviewed from embryos obtained from anonymous donor-egg IVF cycles to determine both the aneuploidy rate and parental source of the genetic error. To measure this, preimplantation genetic screening (PGS) data on embryos were retrospectively collated with parental DNA obtained before IVF for chromosome-specific assessments. This approach permitted mitotic and meiotic copy errors to be differentiated for each chromosome among all embryos tested, thus providing information on the parental source of embryo aneuploidy ( i.e., from the anonymous egg donor vs. sperm source). Results 305 embryos generated for 24 patients who began IVF treatment in 2013. For oocyte donors ( n = 24), mean (±SD) age was 24.0 ± 2.7 years (range = 20-29). For embryos with full chromosomal reporting ( n = 284), euploidy was present in only 133 (46.8%). Considering all embryo chromosomes, the average error rate was 18%. 133 of 151 observed embryo aneuploidies (88.1%) were attributable to an oocyte donor source. Among all aneuploid embryos ( n = 151), chromosomal errors from both genetic parents ( i.e ., oocyte donor and sperm source) were present in 57%. The average correlation coefficient across all pairs of chromosomal abnormalities ( r = 0.60) suggests that chromosomes tend to have multiple and simultaneous errors (complex aneuploidy) even when oocytes from young donors are used. Conclusion These data show that even when young donors provide oocytes for IVF, the probability of embryo aneuploidy remains high. The oocyte donor appears to make an important contribution to embryo aneuploidy even when her age is <30 yrs. If these findings are confirmed with larger, prospective studies, the routine integration of PGS with donor oocyte IVF cycles to identify single euploid embryos for transfer should be considered.

Cancer cells can rapidly adapt and develop resistance to chemotherapy through non-genetic mechanisms involving changes in gene expression programs, in addition to genetic mutations over many generations. One proposed driver of this rapid transcriptional adaptation is alterations in the 3D organization and dynamics of chromatin - the complex that packages and regulates accessibility to the genome inside the nucleus. Chromatin, a dynamic assembly of DNA and associated proteins, behaves as a complex heteropolymer with intricate physical interactions within the nuclear environment. Therefore, beyond the direct influence of epigenetic modifiers on chromatin architecture, factors such as chromatin self-interactions and interactions with nuclear components, including ions, can profoundly impact gene accessibility and transcriptional activity. Adopting a polymer physics perspective facilitates a comprehensive understanding of how chromatin structure and its interactions dictate functional outcomes, such as transcriptional activity and cellular phenotype. Specific chromatin conformations may promote high transcriptional plasticity and activation of pro-survival pathways in response to cytotoxic stress, while other structures restrict this adaptive response resulting in cell death. However, the quantitative rules linking observable multi-scale chromatin structural changes to large-scale transcriptional reprogramming and phenotypic survival decisions remain poorly understood.This dissertation explores the fundamental role of chromatin architecture in dictating transcriptional adaptation to chemotherapy and chemoresistance in cancer. The central hypothesis is that preexisting chromatin conformations permissive to plasticity predispose cells to surviving treatment by enabling rapid transcriptional reprogramming, while restrictive chromatin structures increase cell death by limiting this adaptive response. An integrated experimental and computational approach is employed, including live-cell imaging to map 3D chromatin organization, polymer physics modeling to predict transcriptional plasticity, multi-omics molecular profiling, and pharmacological modulation of chromatin states. Chapter 1 firstly elucidates fundamental aspects of chromatin structure. Developments advanced imaging and quantification techniques to study chromatin architecture with minimal perturbation are described in Chapter 2. Subsequently, in Chapter 3, a computational model is proposed to delineate the mechanistic link between chromatin structure and phenotype, particularly cellular fate decisions driven by transcriptional programs. Furthermore, employing an ovarian cancer model in Chapter 4, this research demonstrates the potential of modulating chromatin organization to constrain phenotypic heterogeneity within cancer stem cell populations, thereby inhibiting the formation of new spheroids and potentially attenuating tumor progression. Chapter 5 concludes with a summary of the work completed in the dissertation and the potential future directions of this project.The findings elucidate key structural determinants of chromatin plasticity across multiple cancer types and their impact on cell survival during cytotoxic stress. Novel chromatin dynamics signatures are identified that predict treatment outcome and chemoresistance. Targeting these chromatin conformations with epigenetic drugs is demonstrated as a strategy to restrict non-genetic adaptation and resensitize resistant cells to therapy. Collectively, this work uncovers fundamental biological principles governing the epigenetic regulation of transcriptional plasticity and adaptation in cancer. These insights have significant implications for developing new therapeutic approaches to overcome treatment resistance by modulating chromatin structure and dynamics.