ChromEMT: Visualizing 3D chromatin structure and compaction n interphase and mitotic cells

By H. D. Ou, Sebastien Phan, Thomas J. Deerinck, Andrea K. Thor, Mark Ellisman, Clodagh C. O’Shea. 2017

800 citations recorded by the source corpus.

A close-up view inside the nucleus The nuclei of human cells contain 2 meters of genomic DNA. How does it all fit? Compaction starts with the DNA wrapping around histone octamers to form nucleosomes, but it is unclear how these further compress into mitotic chromosomes. Ou et al. describe a DNA-labeling method that allows them to visualize chromatin organization in human cells (see the Perspective by Larson and Misteli). They show that chromatin forms flexible chains with diameters between 5 and 24 nm. In mitotic chromosomes, chains bend back on themselves to pack at high density, whereas during interphase, the chromatin chains are more extended. Science, this issue p. eaag0025; see also p. 354 A new technique reveals that chromatin is a disordered 5- to 24-nanometer chain that is packed at different concentration densities according to the cell cycle. INTRODUCTION In human cells, 2 m of DNA are compacted in the nucleus through assembly with histones and other proteins into chromatin structures, megabase three-dimensional (3D) domains, and chromosomes that determine the activity and inheritance of our genomes. The long-standing textbook model is that primary 11-nm DNA–core nucleosome polymers assemble into 30-nm fibers that further fold into 120-nm chromonema, 300- to 700-nm chromatids, and, ultimately, mitotic chromosomes. Further extrapolating from this model, silent heterochromatin is generally depicted as 30- and 120-nm fibers. The hierarchical folding model is based on the in vitro structures formed by purified DNA and nucleosomes and on chromatin fibers observed in permeabilized cells from which other components had been extracted. Unfortunately, there has been no method that enables DNA and chromatin ultrastructure to be visualized and reconstructed unambiguously through large 3D volumes of intact cells. Thus, a remaining question is, what are the local and global 3D chromatin structures in the nucleus that determine the compaction and function of the human genome in interphase cells and mitotic chromosomes? RATIONALE To visualize and reconstruct chromatin ultrastructure and 3D organization across multiple scales in the nucleus, we developed ChromEMT, which combines electron microscopy tomography (EMT) with a labeling method (ChromEM) that selectivity enhances the contrast of DNA. This technique exploits a fluorescent dye that binds to DNA, and upon excitation, catalyzes the deposition of diaminobenzidine polymers on the surface, enabling chromatin to be visualized with OsO4 in EM. Advances in multitilt EMT allow us to reveal the chromatin ultrastructure and 3D packing of DNA in both human interphase cells and mitotic chromosomes. RESULTS ChromEMT enables the ultrastructure of individual chromatin chains, heterochromatin domains, and mitotic chromosomes to be resolved in serial slices and their 3D organization to be visualized as a continuum through large nuclear volumes in situ. ChromEMT stains and detects 30-nm fibers in nuclei purified from hypotonically lysed chicken erythrocytes and treated with MgCl2. However, we do not observe higher-order fibers in human interphase and mitotic cells in situ. Instead, we show that DNA and nucleosomes assemble into disordered chains that have diameters between 5 and 24 nm, with different particle arrangements, densities, and structural conformations. Chromatin has a more extended curvilinear structure in interphase nuclei and collapses into compact loops and interacting arrays in mitotic chromosome scaffolds. To analyze chromatin packing, we create 3D grid maps of chromatin volume concentrations (CVCs) in situ. We find that interphase nuclei have subvolumes with CVCs ranging from 12 to 52% and distinct spatial distribution patterns, whereas mitotic chromosome subvolumes have CVCs >40%. CONCLUSION We conclude that chromatin is a flexible and disordered 5- to 24-nm-diameter granular chain that is packed together at different concentration densities in interphase nuclei and mitotic chromosomes. The overall primary structure of chromatin polymers does not change in mitotic chromosomes, which helps to explain the rapid dynamics of chromatin condensation and how epigenetic interactions and structures can be inherited through cell division. In contrast to rigid fibers that have longer fixed persistence lengths, disordered 5- to 24-nm-diameter chromatin chains are flexible and can bend at various lengths to achieve different levels of compaction and high packing densities. The diversity of chromatin structures is exciting and provides a structural basis for how different combinations of DNA sequences, interactions, linker lengths, histone variants, and modifications can be integrated to fine-tune the function of genomic DNA in the nucleus to specify cell fate. Our data also suggest that the assembly of 3D domains in the nucleus with different chromatin concentrations, rather than higher-order folding, determines the global accessibility and activity of DNA. ChromEMT reveals the in situ chromatin ultrastructure, 3D packing, and organization of DNA. EMT sample volume-DNA-nucleosome chains are black. Chromatin is a structurally disordered 5- to 24-nm granular chain that is packed together at different 3D CVC densities in human interphase nuclei and mitotic chromosomes (red, high density; yellow, medium density; blue, low density). The chromatin structure of DNA determines genome compaction and activity in the nucleus. On the basis of in vitro structures and electron microscopy (EM) studies, the hierarchical model is that 11-nanometer DNA-nucleosome polymers fold into 30- and subsequently into 120- and 300- to 700-nanometer fibers and mitotic chromosomes. To visualize chromatin in situ, we identified a fluorescent dye that stains DNA with an osmiophilic polymer and selectively enhances its contrast in EM. Using ChromEMT (ChromEM tomography), we reveal the ultrastructure and three-dimensional (3D) organization of individual chromatin polymers, megabase domains, and mitotic chromosomes. We show that chromatin is a disordered 5- to 24-nanometer-diameter curvilinear chain that is packed together at different 3D concentration distributions in interphase and mitosis. Chromatin chains have many different particle arrangements and bend at various lengths to achieve structural compaction and high packing densities.

Subfields: Molecular Biology

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

Gene-specific H1 eviction through a transcriptional activator-p300-NAP1-H1 pathway (2019, background): “More recently, advanced microscopic studies (Ricci et al., 2015; Ou et al., 2017) also indicated that transcriptionally inactive genomic regions contain higher H1 densities and more dense chromatin clusters than do transcriptionally inactive regions and, further, identified small, variably sized com”
Chromatin organization by an interplay of loop extrusion and compartmental segregation (2017, background): “As high-resolution (7, 67, 68) and live-cell (69–71) imaging of chromatin is making dramatic progress, such questions may be addressed in the near future.”
Topological polymorphism of nucleosome fibers and folding of chromatin (2021, background): “It would be interesting to compare these results with the superresolution imaging data (11,13).”
Revisit of reconstituted 30-nm nucleosome arrays reveals an ensemble of dynamic structures (2018, methodology): “[46], electron microscopy-assisted nucleosome interaction capture cross-linking [47], in situ spatially correlated DNA cleavage mapping [20] and ChromEMT [48].”
Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome. (2018, positive): “Using estimates of the key parameters of surface tension, molecular interaction energy, and stiffness of euchromatin and heterochromatin (Feric et al., 2016; Ou et al., 2017; Stephens et al., 2017) (see STAR Methods), the model predicts that small droplets can nucleate in both low and high chromatin”
Organization of Chromatin by Intrinsic and Regulated Phase Separation (2019, background): “, 2012) and a lack of higher-order structure in electron tomograms of nuclei (Chen et al., 2016; Ou et al., 2017), calling into question 30 nm fiber-based models of chromatin organization.”