The structure of chromatin plays a vital role in the regulation of gene expression. In this thesis, the mechanical properties of chromatin are investigated by measuring force-extension curves with...Show moreThe structure of chromatin plays a vital role in the regulation of gene expression. In this thesis, the mechanical properties of chromatin are investigated by measuring force-extension curves with Magnetic Tweezers. Two models are re-introduced that accurately describe these curves. Upon adding linker histone H1, a protein that can bind to individual nucleosomes, stabilization of nucleosome unstacking up to 10 pN was observed. Varying the Nucleosome Repeat Length (NRL) of the chromatin fibers with linker histones resulted in different degrees of stabilization: 168NRL fibers have a higher energy barrier for unstacking than 197NRL fibers. This could be caused by increased nucleosome stiffness at lower NRLs. This difference could have an important effect on transcription regulation in vivo.Show less
The accessibility of DNA is regulated by means of dynamic folding and unfolding of chromatin fibers. Quantification of chromatin unfolding is a key in understanding the higher order structure of...Show moreThe accessibility of DNA is regulated by means of dynamic folding and unfolding of chromatin fibers. Quantification of chromatin unfolding is a key in understanding the higher order structure of chromatin. Single molecule force spectroscopy is an ideal tool to study chromatin dynamics. The extension of reconstituted chromatin fibers under force was measured using Magnetic Tweezers. Here I show a model to fit discrete states in chromatin unfolding, and use this model to quantify the influence of linker histone H1 on chromatin fibers. Stepsize analysis revealed that linker histone H1 induces cooperative unstacking of nucleosomes at forces less than 10 pN. Chromatin fibers in presence of linker histone H1 must be stretched $4.5\pm0.8$ nm for two nucleosomes to unstack. This model can be used to quantify natively folded chromatin fibers.Show less
The aim of this study was to computationally resolve nucleosome dynamics and chromatin structure. To achieve this we ran Monte Carlo simulations of a base pair level model of a mononucleosome....Show moreThe aim of this study was to computationally resolve nucleosome dynamics and chromatin structure. To achieve this we ran Monte Carlo simulations of a base pair level model of a mononucleosome. Additionally, we developed a graphical user interface for generating a chromatin structure with realistic linker DNA, which enabled us to calculate linking number and writhe for different chromatin structures. The force extension curve of our simulated mononucleosome shows similar behaviour to force spectroscopy experiments.Show less
