Abstract
Magnetic tweezers form a unique tool to study the topology and mechanical properties of chromatin fibers. Chromatin is a complex of DNA and proteins that folds the DNA in such a way that meter-long stretches of DNA fit into the micron-sized cell nucleus. Moreover, it regulates accessibility of the genome to the cellular replication, transcription, and repair machinery. However, the structure and mechanisms that govern chromatin folding remain poorly understood, despite recent spectacular improvements in high-resolution imaging techniques. Single-molecule force spectroscopy techniques can directly measure both the extension of individual chromatin fragments with nanometer accuracy and the forces involved in the (un)folding of single chromatin fibers. Here, we report detailed methods that allow one to successfully prepare in vitro reconstituted chromatin fibers for use in magnetic tweezers-based force spectroscopy. The higher-order structure of different chromatin fibers can be inferred from fitting a statistical mechanics model to the force-extension data. These methods for quantifying chromatin folding can be extended to study many other processes involving chromatin, such as the epigenetic regulation of transcription.
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Acknowledgments
We are grateful to Nicolaas Hermans, He Meng, Kurt Andresen, Orkide Ordu, and Ineke de Boer for the help in establishing these methods. This work was supported by the Netherlands Organisation for Scientific Research (NWO/OCW), as part of the Frontiers of Nanoscience program, by the NWO-VICI research program, project 680-47-616.
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Kaczmarczyk, A., Brouwer, T.B., Pham, C., Dekker, N.H., van Noort, J. (2018). Probing Chromatin Structure with Magnetic Tweezers. In: Lyubchenko, Y. (eds) Nanoscale Imaging. Methods in Molecular Biology, vol 1814. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8591-3_18
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DOI: https://doi.org/10.1007/978-1-4939-8591-3_18
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