Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy
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This research paper presents a groundbreaking investigation into the real-time dynamics of nucleosome and chromatosome disassembly using high-speed atomic force microscopy (HS-AFM). The authors, led by Bibiana Onoa and Carlos Bustamante, provide a detailed, frame-by-frame visualization of these fundamental chromatin structures as they unwind and dissociate, revealing a multistep asymmetrical process previously only inferred through ensemble and indirect measurements. Their work significantly advances our understanding of how DNA access is regulated through these dynamic processes, and how these processes are altered by the presence of linker histones like H1.
The study's central innovation lies in its application of HS-AFM to visualize the step-by-step changes in nucleosome and chromatosome morphology with sub-second time resolution. This is critical because these structures are highly dynamic, and their disassembly is thought to be a crucial regulator of genome expression and replication. Using this approach, the team is able to track individual nucleosomes and chromatosomes deposited on a mica surface and monitor their transformation from intact complexes to their subnucleosomal products (hexasomes, tetrasomes, and disomes). They carefully controlled the experimental conditions by using magnesium-free buffers supplemented with polyamines to maintain the molecules’ physiological integrity and prevent interactions with the charged surface of the mica. By tracking the changes in the nucleosome core particle (NCP) volume and DNA arm angles in real time, the authors have established a highly detailed picture of their dynamic structural transformations.
One of the key findings of this study is the revelation of the sequential and asymmetrical nature of nucleosome disassembly. The team's work clearly demonstrates that the removal of histone dimers during nucleosome unwrapping is not a symmetrical process. Rather, they show that the H2A-H2B heterodimer located on the rigid side of the 601 nucleosome positioning sequence (NPS) dissociates first, causing a significant change in the short DNA arm angle and a slight decrease in the histone core volume. This initial dimer dissociation is followed by the ejection of the second H2A-H2B heterodimer on the flexible side of the NPS, which prompts further changes in the long DNA arm angle and additional volume loss. This finding strongly corroborates models based on earlier studies by sm-FRET, time-resolved SAXS, and molecular dynamics.
Further analysis of the real-time data revealed that the formation of a tetrasome, after the dissociation of the two H2A-H2B heterodimers, marks a significant change in the disassembly pathway, slowing the rate of disassembly considerably. The tetrasome, consisting of the (H3-H4)2 complex, was found to be particularly stable. This observation provides a compelling reason for the longevity of tetrasomes in vivo, as has been previously documented in other studies involving RNA polymerase activity. The team also reports a dynamic, constrained diffusion of the tetramer along the dyad of the NPS, suggesting that it can slide along the DNA, potentially allowing it to find an optimal position for subsequent nucleosome assembly and function. This dynamic tetramer behavior may also be critical to how nucleosomes are assembled and positioned on the genome.
The presence of linker histone H1 was also found to dramatically affect the disassembly of the nucleosome. When H1 is present, it transforms the nucleosome into a more compact structure, a chromatosome, by stabilizing the linker DNA regions at the entry-exit site. The binding of H1 dramatically increased the lifetime of the complex before the first heterodimer dissociation, effectively slowing down disassembly by a factor of two. This stabilization of DNA also constrained DNA unwrapping and, thus, altered the pathway of heterodimer release. Interestingly, when the first dimer is released from a chromatosome, the linker histone frequently docks reversibly back into the core before a second dissociation step occurs. This rebinding of H1 is not observed in nucleosomes and further demonstrates the impact of the linker histone. Finally, the dissociation of chromatosomes, like that of nucleosomes, is ultimately a sequential and asymmetrical process that leaves a tetrasome as its most stable intermediate.
To analyze the dynamic data, the researchers implemented a neural network for automated segmentation of the molecules. By training this network with manually segmented nucleosomes and PANS, the team was able to differentiate between the protein and DNA components, thus tracking the NCP volume changes and DNA arm movements automatically. Their approach was crucial for the high-throughput analysis required by the dynamic data obtained by HS-AFM and represents a powerful tool for the analysis of such data. It demonstrates how a combination of machine learning and innovative microscopy can be leveraged to advance our understanding of biological structures.
The methodology employed by Onoa and colleagues is noteworthy for its use of purified, well-defined nucleosomes and chromatin components. They carefully reconstituted these molecules and characterized their purity through gel electrophoresis, reducing the possibility of artifacts that can arise from the use of less defined samples. They also performed control experiments, like crosslinking the molecules to prevent unwrapping, and demonstrated that the observed unwrapping is driven by intrinsic dynamics, rather than induced by interactions with the AFM tip or surface. This careful control and characterization of the experimental system is vital for making robust and reproducible observations.
The implications of this research are far-reaching for our understanding of genome regulation. The work underscores the dynamic and asymmetrical nature of nucleosome disassembly and how this is affected by the linker histone H1. These insights suggest that the dynamics of nucleosome disassembly could play a key role in regulating gene expression. The authors suggest that the polarity of the unwrapping as well as the timing of the dimer release are important determinants of transcriptional access to the DNA. The finding that the tetrasome is a particularly stable intermediate that can diffuse along the DNA adds a new layer of complexity and has implications for both nucleosome assembly and its role in transcription elongation.
Moreover, the study's use of HS-AFM opens new avenues for research in this field. The ability to directly visualize the structural dynamics of nucleosomes and their disassembled products in real time provides a powerful tool for investigating the effects of different protein interactions, epigenetic modifications, and enzyme activities on nucleosome structure and stability. This paper provides a firm foundation for further exploration into the complex and dynamic world of chromatin.
In conclusion, this study provides a significant contribution to the field of chromatin biology by leveraging the strengths of HS-AFM, an innovative data processing pipeline, and well-defined nucleosome and chromatin components. The team's findings demonstrate the sequential, asymmetrical nature of nucleosome disassembly, the importance of tetrasomes in the chromatin landscape, and the profound impact of linker histone H1. The methodologies, analysis, and interpretations of this research are a significant advance in our understanding of chromatin structure and function and will be invaluable for exploring the dynamic nature of this fundamental biological structure in the future.
https://pubs.acs.org/doi/10.1021/acscentsci.3c00735
