Magnetic tweezers based force spectroscopy studies of the structure and dynamics of nucleosomes and chromatin

Kruithof, M.C. (2009)

Doctoral Thesis

Animals and plants are build from a large number of cells. These cells continuously respond to signals from outside and inside the cell by producing various kinds of proteins. The blueprints of these proteins are stored in genes. The genes, in cells with a nucleus, are carried in chromosomes: threadlike structures in the nucleus of a cell that become visible when the cell, upon dividing, condenses these structures. Chromosomes consist of roughly two parts: proteins, that take care of the condensation and DNA, carrying the genetic information of the cell. Without this condensation, the DNA in a human cell would never fit into the nucleus. During a cell division, DNA is compacted even more. The condensation has to be done in an orderly fashion so that the chromosomes can be replicated correctly at each cell division. Besides the compaction, the DNA still needs to be accessible for the expression of genes. The activity of genes can even be controlled by regulation of the DNA compaction. For a complete understanding of the regulation of DNA compaction, we need to understand, at molecular detail, not only the structure but also the dynamics of the compaction of DNA. At the first level of compaction, DNA winds around specific proteins, called histones. The DNA-histon complex is called a nucleosome. Another species of histone proteins, called linker histones are known to constrict the DNA exiting the nucleosome, thereby stabilizing the structure of the nucleosome. Under physiological conditions, arrays of nucleosomes fold into compact fibers called chromatin fibers. The transient structure of nucleosomes and the interaction between nucleosomes in a chromatin fiber, plays an important role in the compaction of DNA. We chose to use force spectroscopy, because this technique makes it possible to study the structure and dynamics of nucleosomes at the level of single molecules. In chapter 2 we introduced a simple method for dynamic force spectroscopy using magnetic tweezers. This method allows application of sub-piconewton force on single molecules, by calibration of the applied force from the distance between a pair of magnets and a magnetic sphere, which is used to apply a force to a molecule. Initial dynamic force spectroscopy experiments on DNA molecules revealed a large hysteresis in the force-extension curve. This hysteresis was caused by viscous drag on the magnetic bead making it impossible to measure the weak interactions between DNA and nucleosomes. Smaller beads decreased this hysteresis sufficiently to reveal intra-molecular interactions at sub-piconewton forces. Compared to typical quasi-static force spectroscopy our method is significantly faster, allowing the real time study of transient structures and reaction intermediates. As a proof of principle nucleosome-nucleosome interactions on a sub-saturated chromatin fiber were analyzed. In chapter 3 we investigated the Brownian fluctuations of the magnetic sphere in a magnetic tweezers experiment. We measured the force induced unwrapping of DNA from a single nucleosome. We showed that hidden Markov analysis, adopted for the non-linear force-extension of DNA, can readily resolve unwrapping events that are significantly smaller than the Brownian fluctuations. The probability distribution of the height of the magnetic bead was used to accurately resolve small changes in contour length and persistence length of a DNA molecule containing a nucleosome. The latter is shown to be directly related to the DNA bending angle of the complex. The adapted hidden Markov analysis can be used for any transient DNA-protein complex and provides a robust method for the investigation of these transient events. In chapter 4 we used magnetic tweezers to probe the mechanical properties of a single, well-defined array of 25 nucleosomes folded into a chromatin fiber. We found that the fiber stretched linearly like a Hookian spring to more than three times its starting length at forces up to 4\mbox{ pN}. This unexpected large extension points to a solenoid as the underlying topology of the chromatin fiber. Surprisingly, linker histones do not affect the length or stiffness of the fibers. They do stabilize the fiber at forces up to 7\mbox{ pN}. Fibers with a nucleosome repeat length of 167 basepairs instead of 197 basepairs are significantly stiffer, consistent with a two-start helical arrangement. The extensive thermal breathing of the chromatin fiber that is a consequence of the observed high compliance provides a structural basis for understanding the balance between compaction of DNA to fit into the cell core and the transparency of DNA to allow proteins to access the genetic information of the cell. In chapter 5 we investigated the unexpected difference in the force needed for the unwrapping of the first turn and unwrapping of the second turn of nucleosomes in experiments on single nucleosomes and nucleosomes in a fiber. The forces needed to unwrap a single nucleosome were much smaller, 3 pN for the first turn and 6 pN for the second turn, than those for a nucleosome in a fiber, 6 pN and 18 pN respectively. We modeled a nucleosome-DNA-bead system, used in force spectroscopy experiments, as spheres and springs. We found that the thermal fluctuations of neighbouring nucleosomes stabilized the nucleosome thereby increasing the unwrapping force for a nucleosome in a fiber. This effect shows that results obtained for single nucleosomes cannot simply be extrapolated to a system containing multiple nucleosomes.

• Chromatin
• Dynamic force spectroscopy
• Force spectroscopy
• Hidden markov
• Magnetic tweezers
• Nucleosomes
• Single molecules

• All authors: Kruithof, M.C.
• Supervisor: Schmidt, T.
• Co-supervisor: Noort, J. van
• Qualification: Doctor (dr.)
• Awarding Institution: Faculty of Science, Leiden University
• Date: 2009-10-01
• ISBN (print): 9789085930570
• Sponsorship: NWO