Mechanisms and roles of chromatin organization – decipher the epigenetic code

With this method, the labs of Johannes Stigler (LMU Munich), Luis Aragon (MRC-IMS, London), and Shixin Liu (The Rockefeller University, NY) were able to identify unique binding and mobility behavior of SMC5/6 on ss- vs ds-DNA and could identify DNA fork structures as preferred binding location, emphasizing SMCs’ critical role in DNA replication and repair (see figure).

The real-time single-molecule imaging capabilities of the C-Trap also enabled Frank Uhlmann (The Francis Crick Institute, London) and his collaborators to directly observe condensin interacting with multiple dsDNA molecules, a mechanism that had earlier been identified for cohesin using a similar C-Trap assay.

In summary, dynamic single-molecule (DSM) microscopy is key to quantifying

• binding rates and location of SMC complexes on different DNA structures
• dynamic movement of SMC complexes on DNA and looping/cross-linking activity
• influence of additional factors (ATP, small molecules, other DNA-binding proteins) on SMC kinetics and dynamics

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Use the force: By applying tension on cohesin-looped DNA, Luis Aragon and collaborators were able to quantify loop size as well as mechanical stability of the cohesin-mediated cross-links.

Furthermore, tension can also be directly applied to SMC complexes (or subunits) to quantify their conformational dynamics under force. By doing so, Johannes Stigler and colleagues identified structures and intermediate states in the coiled-coil region of SMCs that are critical for dynamic folding of the complex. For more details on the assay, see our protein folding page.

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Read more:

• Chang et al. Nature Communications (2022)
• Tanasie et al. Cell Reports (2022)
• Gutierrez-Escribano et al. Science Advances (2019)
• Tang et al Molecular Cell (2023)
• Freitag et al. Biophysical Journal (2022)

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The groups of Taekjip Ha (Harvard University), Carl Wu (Johns Hopkins University Baltimore) and Shixin Liu (The Rockefeller University) have extensively used LUMICKS’ C-Trap to unravel details of remodeler target search and to discover hitherto unknown nucleosome remodeling capabilities of origin recognition complexes (ORCs), respectively.

Use the force: The optical traps of the C-Trap allow the application of well-defined mechanical tension on a single DNA molecule, mimicking the case of incomplete sister chromatid segregation or other mechanically challenging scenarios DNA is regularly facing.

By doing so, the groups of Erwin Petermann and Gijs Wuite (VU Amsterdam) in collaboration with Ian Hickson (University of Copenhagen) discovered an unknown role of the checkpoint helicase PICH: On DNA under tension, PICH shows nucleosome unwrapping and histone transporting activities, as directly observed with correlative dynamic single-molecule microscopy (see figure).

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Read more:

• Kim et al. eLife (2024)
• Li et al. PNAS (2022)
• Spakman et al. Nature Communications (2022)

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By tethering arrays of nucleosomes between two optical traps, you gain access to quantitative information about the mechanical stability of individual nucleosomes as well as possible cross-linking interactions.

Additional factors like histone-modifying enzymes or nucleosome cross-linkers are quickly added via the integrated microfluidics and their interaction with the DNA/nucleosomes can be observed in real-time with single-molecule fluorescence microscopy.

The effects these factors have on nucleosome stability and cross-linking can be directly assessed by increasing the tension on the DNA substrate (see figure): Depending on the modification, the accessibility of DNA can be increased (destabilization, lower force required) or decreased (stabilization, higher force required).

In summary, with the dynamic single-molecule (DSM) approach you gain quantitative access to the

• location, duration and sequential order of modifier-nucleosome interactions
• mechanical stability of native and modified nucleosomes
• strength of nucleosome-nucleosome interactions mediated by cross-linking factors

Amongst other labs, the groups of Mark Williams (Northeastern University Boston) and Shixin Liu (The Rockefeller University New York) used this method successfully to unravel the impact of histone chaperones on nucleosome (dis)assembly, and Polycomb complex-mediated nucleosome cross-linking, respectively.

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Read more:

• McCauley et al. Cell Reports (2022)
• Leicher et al. PNAS (2020)

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