Hi-C and derivative techniques, although effective at detecting longer-range interactions, suffer from high noise below 5 to 100 kb, depending on experimental factors such as read depth and restriction enzymes ( 17). To characterize these details of chromatin organization and understand their relation to gene transcription at all length scales, it is necessary to overcome several fundamental limitations of existing techniques. Despite the advancements in visualizing nanocompartments, several critical open questions remain, including how the chromatin chain packs into these and other higher-order structures, the mechanisms of formation and maintenance of chromatin conformation in live cells, and the connection between chromatin conformation, gene loci connectivity, and transcription processes. ( 14) elucidated the coherent dynamics of chromatin domains in live cells using SR imaging and single-nucleosome tracking. Multiple independent studies have reported the existence of TAD-like chromatin nanocompartments using SR microscopies ( 13– 16). Recently, the development of super-resolution (SR) microscopies, including stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy, in combination with labeling methods, such as fluorescence in situ hybridization (FISH) and DNA point accumulation in nanoscale topology, has allowed for direct comparison between microscopy and Hi-C techniques. ( 8) found that DNA and nucleosomes assemble into disordered chains, with diameters varying between 5 and 24 nm, which themselves pack at various densities within the nucleus. One such work used a novel imaging technique, chromatin electron tomography (ChromEMT), to interrogate chromatin ultrastructure down to the level of single nucleosomes ( 8). Previously, the primary 11-nm fiber was thought to aggregate into a thicker 30-nm chromatin fiber, but this textbook view has been challenged by several recent studies ( 6, 7). The basic units of chromatin are nucleosomes, which are connected by linker DNA to form a “beads-on-a-string” chromatin fiber. However, the precise conformation of chromatin and its relationship with transcription, a direct determinant of cellular phenotype, remain contested. Large-scale alterations in chromatin structure are associated with cancer, numerous neurological and autoimmune disorders, and other complex diseases ( 4, 5). Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.ĭynamic, three-dimensional (3D) chromatin organization plays an important role in regulating a vast number of cellular processes, including cell type–specific gene expression and lineage commitment ( 1– 3). The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. 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. 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. 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. Almassalha, and Vadim Backman Show FewerĮxtending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. Chandler, The-Quyen Nguyen, Reiner Bleher, Juan J. Pujadas, Surbhi Jain, George Esteve, John E. Bauer, Xiang Zhou, Vasundhara Agrawal, Emily M. Virk, Aya Eid, Wenli Wu, Jane Frederick, David VanDerway, … Show All …, Scott Gladstein, Kai Huang, Anne R.
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