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1. Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

   https://doi.org/10.1038/s41467-020-19876-6  

   Cremer, Marion; Brandstetter, Katharina; Maiser, Andreas; Rao, Suhas S.; Schmid, Volker J.; Guirao-Ortiz, Miguel; Mitra, Namita; Mamberti, Stefania; Klein, Kyle N.; Gilbert, David M.; et al (December 2020, Nature Communications)

   null (Ed.)

   Abstract Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity. 

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

   https://doi.org/10.1126/sciadv.adq6652  

   Almassalha, Luay M; Carignano, Marcelo; Liwag, Emily Pujadas; Li, Wing Shun; Gong, Ruyi; Acosta, Nicolas; Dunton, Cody L; Gonzalez, Paola Carrillo; Carter, Lucas M; Kakkaramadam, Rivaan; et al (January 2025, Science Advances)

   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. 

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3. Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization

   https://doi.org/10.1038/s41467-024-48698-z  

   Kant, Aayush; Guo, Zixian; Vinayak, Vinayak; Neguembor, Maria Victoria; Li, Wing Shun; Agrawal, Vasundhara; Pujadas, Emily; Almassalha, Luay; Backman, Vadim; Lakadamyali, Melike; et al (December 2024, Nature Communications)

   Abstract In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets. 

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4. Analysis of three-dimensional chromatin packing domains by chromatin scanning transmission electron microscopy (ChromSTEM)

   https://doi.org/10.1038/s41598-022-16028-2  

   Li, Yue; Agrawal, Vasundhara; Virk, Ranya K.; Roth, Eric; Li, Wing Shun; Eshein, Adam; Frederick, Jane; Huang, Kai; Almassalha, Luay; Bleher, Reiner; et al (December 2022, Scientific Reports)

   Abstract 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. 

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5. Loops and the activity of loop extrusion factors constrain chromatin dynamics

   https://doi.org/10.1091/mbc.E23-04-0119  

   Bailey, Mary Lou; Surovtsev, Ivan; Williams, Jessica F.; Yan, Hao; Yuan, Tianyu; Li, Kevin; Duseau, Katherine; Mochrie, Simon G.; King, Megan C. (July 2023, Molecular Biology of the Cell)

   Bloom, Kerry (Ed.)

   The chromosomes—DNA polymers and their binding proteins—are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number of loops or residence times of loop extrusion factors influence the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taking the data together, we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops. 

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