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
Apelt, K.; Lans, H.; Scharer, O.D.; Luijsterburg, M.S. 2021
Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair... Show moreGlobal genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes. Show less
Grosch, M.; Ittermann, S.; Shaposhnikov, D.; Drukker, M.E. 2020
Membrane-free intracellular biocondensates are enclosures of proteins and nucleic acids that form by phase separation. Extensive ensembles of nuclear "membraneless organelles" indicate their... Show moreMembrane-free intracellular biocondensates are enclosures of proteins and nucleic acids that form by phase separation. Extensive ensembles of nuclear "membraneless organelles" indicate their involvement in genome regulation. Indeed, nuclear bodies have been linked to regulation of gene expression by formation of condensates made of chromatin and RNA processing factors. Important questions pertain to the involvement of membraneless organelles in determining cell identity through their cell-type-specific composition and function. Paraspeckles provide a prism to these questions because they exhibit striking cell-type-specific patterns and since they are crucial in embryogenesis. Here, we outline known interactions between paraspeckles and chromatin, and postulate how such interactions may be important in regulation of cell fate transitions. Moreover, we propose long non-coding RNAs (lncRNAs) as candidates for similar regulation because many form foci that resemble biocondensates and exhibit dynamic patterns during differentiation. Finally, we outline approaches that could ascertain how chromatin-associated membraneless organelles regulate cellular differentiation. 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
Background DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes... Show moreBackground DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes that affect DNA methylation patterns in blood using large-scale population genomics data. Results By employing genetic instruments as causal anchors, we establish directed associations between gene expression and distant DNA methylation levels, while ensuring specificity of the associations by correcting for linkage disequilibrium and pleiotropy among neighboring genes. The identified genes are enriched for transcription factors, of which many consistently increased or decreased DNA methylation levels at multiple CpG sites. In addition, we show that a substantial number of transcription factors affected DNA methylation at their experimentally determined binding sites. We also observe genes encoding proteins with heterogenous functions that have widespread effects on DNA methylation, e.g.,NFKBIE,CDCA7(L), andNLRC5, and for several examples, we suggest plausible mechanisms underlying their effect on DNA methylation. Conclusion We report hundreds of genes that affect DNA methylation and provide key insights in the principles underlying epigenetic regulation. Show less
Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate... Show moreDifferentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The Nucleosome Remodelling and Deacetylation (NuRD) complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. Here we find that while NuRD activity is required for lineage commitment from primed pluripotency in both human and mouse cells, the nature of this requirement is surprisingly different. While mouse embryonic stem cells (mESC) and epiblast stem cells (mEpiSC) require NuRD to maintain an appropriate differentiation trajectory as judged by gene expression profiling, human induced pluripotent stem cells (hiPSC) lacking NuRD fail to even initiate these trajectories. Further, while NuRD activity is dispensable for self-renewal of mESCs and mEpiSCs, hiPSCs require NuRD to maintain a stable self-renewing state. These studies reveal that failure to properly fine-tune gene expression and/or to reduce transcriptional noise through the action of a highly conserved chromatin remodeller can have different consequences in human and mouse pluripotent stem cells. 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
Spits, M.; Janssen, L.J.; Voortman, L.M.; Kooij, R.; Neefjes, A.C.M.; Ovaa, H.; Neefjes, J. 2019
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
Mason, A.G.; Slieker, R.C.; Balog, J.; Lemmers, R.J.L.F.; Wong, C.J.; Yao, Z.Z.; ... ; Maarel, S.M. van der 2017
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
Benard, A.; Janssen, C.M.; Elsen, P.J. van den; Eggermond, M.C.J.A. van; Hoon, D.S.B.; Velde, C.J.H. van de; Kuppen, P.J.K. 2014
Since the discovery of the right-handed helical structure of DNA, 61 years have passed. The DNA molecule, which encodes genetic information, is also found twisted into coils. This extra twist of... Show moreSince the discovery of the right-handed helical structure of DNA, 61 years have passed. The DNA molecule, which encodes genetic information, is also found twisted into coils. This extra twist of the helical structure, called supercoiling, plays important roles in both DNA compaction and gene regulation. The DNA in eukaryotic cells is packaged into chromatin. Using single-molecule force spectroscopy, I resolved force/torque induced structural changes of DNA and chromatin fibers. I showed that the structural changes of chromatin fibers can be described by four conformations. I showed for the first time the folding and unfolding of a chromatin fiber under torsion. Th e anisotropic response of chromatin fibers to supercoiling reflects its leftŸ-handed chirality. These findings give a detailed structural insight of a supercoiled chromatin fiber, yielding a better understanding of the response of chromatin during transcription Show less
Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the... Show moreUnderstanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of individual chromatin proteins on DNA. Characterization of the architectural properties of these proteins is essential to understand how they shape and modulate the archaeal genome. This thesis describes the biophysical characterization of several chromatin proteins from the crenarchaeal model organism Sulfolobus solfataricus: Cren7, Sul7, Alba and Sso10a. The architectural properties of these proteins resemble those of their bacterial counterparts, suggesting that they could play a similar role in chromatin organization and global gene regulation. 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
