In this thesis I describe the results of Pulsed Interleaved Excitation and Fluorescence (Cross) Correlation Spectroscopy (PIE-F(C)CS) combined with single-pair Förster Resonance Energy Transfer ... Show moreIn this thesis I describe the results of Pulsed Interleaved Excitation and Fluorescence (Cross) Correlation Spectroscopy (PIE-F(C)CS) combined with single-pair Förster Resonance Energy Transfer (spFRET) used to study dynamics in single nucleosomes, which depends on subtle differences in the length of DNA ends, DNA sequence, histone variants and specific and non-specific protein interactions. This technique, which can resolve distances between two fluorophores of only a few nanometers, is an excellent technique to monitor changes in nucleosomal compaction, as the nucleosome is only ten nanometers in diameter. In combination with F(C)CS and PIE, spFRET makes it possible to monitor conformational dynamics on a timescale of micro- to milliseconds. Show less
Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic repression of the D4Z4 repeat resulting in misexpression of the repeat-encoded DUX4 gene in skeletal muscle. Two... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic repression of the D4Z4 repeat resulting in misexpression of the repeat-encoded DUX4 gene in skeletal muscle. Two mechanisms are known to drive this D4Z4 epigenetic dysregulation: a contraction of the D4Z4 repeat or mutations in DNMT3B or SMCHD1, both epigenetic regulators of the repeat that are responsible for the establishment or maintenance of the repeat’s epigenetic repressive state in somatic cells. However, the aforementioned (epi)genetic changes lead to FSHD only if the individual also has a disease-permissive D4Z4 allele which allows for stable DUX4 expression in skeletal muscle. This disease permissivity of D4Z4 alleles has been attributed to the presence of a DUX4 polyadenylation signal adjacent to the D4Z4 repeat which is used for transcription termination. Despite knowing the root cause of FSHD, to date, there is no curative therapy available for FSHD and in some cases, the genetic etiology of the disease remains unknown. In this thesis, we identified a new FSHD disease gene called LRIF1 and performed its follow-up functional studies in human somatic cells and mouse embryonic stem cells. In addition, we also pursued a new targeted gene therapy for FSHD by employing CRISPR-based mutagenesis of the DUX4 polyadenylation signal. Show less
Epigenetic regulation of gene expression by chromatin modifiers is one of the fundamental cellular processes that allow the different cell types in the body to develop from the totipotent embryonic... Show moreEpigenetic regulation of gene expression by chromatin modifiers is one of the fundamental cellular processes that allow the different cell types in the body to develop from the totipotent embryonic stem cells. However, when this epigenetic control mechanism becomes compromised, such as by mutations in chromatin modifiers, it can lead to the development of disease. An example of such epigenetic disease is facioscapulohumeral muscular dystrophy (FSHD), in which the chromatin structure of the D4Z4 macrosatellite repeat is compromised. The loss of a repressive D4Z4 chromatin structure either by contraction of the repeat to a size of 1-10 D4Z4 units (FSHD1), or by mutations in D4Z4 chromatin repressors such as SMCHD1 (FSHD2), results in inappropriate expression of the DUX4 gene from the repeat in skeletal muscle, which is considered the root cause of FSHD.In FSHD, DUX4 expression causes apoptosis, leading to muscle wasting in the patient. In this thesis, we studied the functionality of SMCHD1, and aimed to understand the DUX4 repressive processes in which SMCHD1 is involved. Furthermore, we gathered information on the different roles that SMCHD1 fulfills, such as X-chromosome inactivation in female cells and telomere maintenance. Show less
Melanoma is the most aggressive and lethal type of skin cancer since it has the ability to spread to other organs in the body making it harder to control the disease.In this thesis, we aim to... Show moreMelanoma is the most aggressive and lethal type of skin cancer since it has the ability to spread to other organs in the body making it harder to control the disease.In this thesis, we aim to explore the degree to which epigenetics play a role in melanoma, namely, inherited and acquired epigenetic alterations in melanoma susceptibility and development. Show less
In human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional... Show moreIn human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional proteins. Such plasticity of the genome is maintained by dynamic folding and unfolding of DNA-protein spools called nucleosomes. It is unclear, however, how this process is controlled when multiple nucleosomes stack on top of each other and form compact chromatin fibers. This is particularly important since nucleosomes are rarely present in isolation inside a densely packed cell nucleus. Therefore, the aim of this thesis was to increase the understanding of the chromatin fiber structure and its dynamics. Knowing these details would provide many new insights into the mechanisms of gene expression (epigenetic regulation) which, upon malfunction, may cause severe diseases. The presented work consists of an experimental approach involving the application of single-molecule force spectroscopy, and makes use of theoretical modelling based on statistical mechanics. By using magnetic tweezers, we stretched and twisted individual chromatin fibers reconstituted in vitro in order to unfold its nucleosomes. These studies show that folding of nucleosomes into chromatin fibers opens up a plethora of regulatory pathways for controlling the level of DNA organization in cells. Show less
In this thesis two diseases that share a common feature of hypomethylation of repetitive DNA are studied: facioscapulohumeral muscular dystrophy (FSHD) and immunodeficiency, centromeric... Show moreIn this thesis two diseases that share a common feature of hypomethylation of repetitive DNA are studied: facioscapulohumeral muscular dystrophy (FSHD) and immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. In FSHD there is hypomethylation of the macrosatellite repeat D4Z4 and the associated DUX4 gene, which is caused by a repeat contraction and/or variants in chromatin modifiers essential for a repressive D4Z4 chromatin structure in somatic cells. In ICF there is hypomethylation of centromeric repeats, which is caused by recessive variants in one of four ICF genes, of which two are established chromatin modifiers. In this thesis, the mutation spectrum of FSHD and ICF has been expanded. The SMCHD1 mutation spectrum in FSHD2 has been expanded with the discovery of exonic SMCHD1 variants, intronic SMCHD1 variants, and whole SMCHD1 gene deletions. In addition, we identified heterozygous variants in a new FSHD2 gene, DNMT3B, in two FSHD2 families. For ICF syndrome we expanded the mutation spectrum in the two most common ICF genes, DNMT3B and ZBTB24. Show less
A large part of the human genome consists of repetitive DNA. In this thesis two human diseases have been studied in which deregulation of repetitive DNA is a central feature: facioscapulohumeral... Show moreA large part of the human genome consists of repetitive DNA. In this thesis two human diseases have been studied in which deregulation of repetitive DNA is a central feature: facioscapulohumeral muscular dystrophy (FSHD) and immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. FSHD is caused by the misexression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded in the D4Z4 repeat array and is silenced in healthy somatic tissues. In this thesis, several aspects of the epigenetic deregulation of DUX4 in FSHD are described. We have analysed possible correlations between disease severity and epigenetic organization of the D4Z4 repeat. Next we showed that cellular ageing results in deregulation of genomic regions like D4Z4. Moreover, we show that SMCHD1 is the main epigenetic repressor of DUX4 in somatic cells. We next showed that DUX4 misexpression results in the activation of an FSHD candidate gene, FRG2. Finally, we report the generation of a transgenic mouse model for FSHD. The disease mechanism of ICF syndrome remains to be elucidated. However, in this thesis we identify two new ICF disease genes. We highlight a role for all four known ICF genes in repressing repetitive DNA, suggesting functional convergence of these genes. Show less
Polymers are the main building blocks of many biological systems, and thus polymer models are important tools for our understanding. One such biological system is the large scale organisation of... Show morePolymers are the main building blocks of many biological systems, and thus polymer models are important tools for our understanding. One such biological system is the large scale organisation of chromatin. A key question here, is how during cell division the chromosomes can separate without entanglement and knotting. One proposal is that this achieved by a specific spatial organisation of the chromosomes, known as the "fractal globule". Using Monte Carlo simulations, we found that fractal globules are unstable and thus cannot represent the biological system without further ingredients. Another proposal is that topological effects cause spatial separation of the chromosomes. These topological effects can be studied using simulations of nonconcatenated ring polymers. Using a compute device called the Graphics Processing Unit, very detailed and long simulations were carried out. From these a picture emerged in which ring polymers behave much slower than was found in previous studies. A second biological system studied here is the folded state of the protein. This is modeled by the Hamiltonian walk. Here, instead of simulations, we exactly enumerated all Hamiltonian walks of the 4x4x4 cube. Interestingly, simulations show that for larger systems many more walks exist than previously estimated. Show less
The influence of temperature on various elastic properties of DNA is analyzed close to elastic instabilities. The buckling transition under compression is interpreted as decreasing. Under torsion a... Show moreThe influence of temperature on various elastic properties of DNA is analyzed close to elastic instabilities. The buckling transition under compression is interpreted as decreasing. Under torsion a first order phase transition is described ending in an important multi-plectoneme phase that changes to a line of critical points in the infinite chain limit. Show less
In eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is... Show moreIn eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is connected to the adjacent nucleosomes with linker DNA. The folding of chromatin fibers effectively increases the compaction of genomic DNA, but it remains accessible for enzymatic reactions. This apparent paradox motivates a detailed study of the dynamics of chromatin. A structural model at the molecular level will shed light on how cells regulate the compaction and dynamics of genomic DNA. This thesis presents the results of an experimental study on the dynamics of chromatin higher-order folding. Using magnetic tweezers, we observed force-dependent structural changes within chromatin fibers at the single nucleosome level. Show less
In this thesis we attempt to provide a better understanding of the principles that underlie the spatial dynamic organization of the cell nucleus. Chapter 1 reviews the current status of knowledge... Show moreIn this thesis we attempt to provide a better understanding of the principles that underlie the spatial dynamic organization of the cell nucleus. Chapter 1 reviews the current status of knowledge about the structural and functional organization of the cell nucleus. In chapter 2, the development of a computer program is described that has been designed to track the 2D and 3D motion of objects in the nucleus of living cells. In chapter 3, evi-dence is provided for the existence of a nuclear matrix structure that is composed of lamin proteins, emerin and actin. By analyzing the dynamics of telomeres in nuclei of cells showing reduced levels of lamin expression, it is investigated whether telomeres anchor to an inner nuclear lamina structure. In chapter 4 the de novo formation of PML nuclear bodies is described. Using live cell imaging and immunocytochemistry it is dem-onstrated that telomeres play a role in the de novo formation of PML bodies. In chapter 5 it is investigated whether nuclear bodies are associated with chromatin in the cell nucleus. After treating cells with DNA alkylating agent MMS, the dynamics of PML bodies, Cajal bodies and speckles has been analyzed relative to chromatin in the 3D space of the cell nucleus Show less
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... Show moreAnimals 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. Show less
