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A novel perspective on the in vivo chromatin landscape of Saccharomyces cerevisiae
Get PDFChromatin is the defining feature of the eukaryotic genome and is essential for survival. The basic units of chromatin are abundantly present and are referred to as nucleosomes. These nucleosomes are often described and visualized as ‘beads on a string’. However, unlike beads on a string, nucleosomes are not randomly scattered across the chromatin, but are generally well positioned in a stereotypical pattern. This pattern is characterized by a nucleosome free region (NFR), followed by a regular pattern – or array – of nucleosomes which are typically phased relative to a defined genomic location. The organization of nucleosomes allows for controlled gene expression and is therefore regarded as a key regulatory element. A major influence on the position of nucleosomes are ATP dependent chromatin remodelers. These remodelers may work synergistically or antagonistically and often take redundant roles in positioning nucleosomes throughout the chromatin landscape. Mutations or deletions of these remodelers can lead to genome instability, loss of array regularity or diminished phasing, thus increasing heterogeneity in the chromatin landscape. Besides remodelers, other minor factors such as the inherent DNA sequence and general regulatory factors (GRFs) influence the chromatin landscape. Moreover, the chromatin landscape across independent (healthy) cells and genomic locations may be altered or vary significantly as a result of the cell cycle stage or genome replication. Due to the plethora of different factors that can be of influence, it is difficult to dissect how the chromatin landscape is shaped.
In this thesis, we investigate generation and heterogeneity of the chromatin landscape in S. cerevisiae. To this end, we initially developed a novel sequencing approach that allows us to probe the location of nucleosomes on a single chromatin fiber. We show that this approach provides several unique insights allowing us to determine cell-to-cell heterogeneity and which factors attribute to array regularity. Moreover, we provide a detailed description of experiment procedures and analyses that result in optimal reproducibility, accuracy and throughput.
We applied our novel approach to initially investigate the rDNA loci in vivo and utilized our high throughput and accuracy to scrutinize previous observations on these loci. Our results find a similar dichotomy in the chromatin landscape, but do not agree with the transcriptionally independent character proposed previously. As our approach could uniquely detect the heterogeneity of individual chromatin fibers, we investigated the underlying regularity of arrays that would otherwise be classified as irregular using traditional techniques. We find that despite a lack of phasing, nearly all arrays display a high regularity in wild type cells. Similarly, previous findings suggested that lack of ISWI and CHD remodelers would result in unphased arrays, but could not definitively provide evidence for a lack of regularity. Here, we show direct evidence that deletion of these remodelers results in unphased and irregular nucleosomal arrays. We further investigate in vitro findings suggesting a ‘clamping’ model in which remodeler activity results in a density independent array spacing. Comparison of computational modeling with our in vivo findings do not support this model. In support of previous findings by our lab, we identify that INO80 is a bona fide spacing remodeler. We further provide preliminary evidence that this novel approach can be used to detect strain variants in order to sensitively detect global changes in nucleosome occupancy.
In the final chapter of this thesis, we investigate the spacing mechanism of the ISWI remodeler utilizing our sequencing approach in combination with an in vitro chromatin array. Previous findings in our lab have demonstrated the ability to investigate the kinetics of the remodeler using this array in combination with a restriction based assay. However, these results do not provide details on the remodeling process and how nucleosomes are moved on a single fiber. To this end, we first designed a novel DNA template containing multiple Widom 601 sequences and linkers optimized for our sequencing approach. These optimizations would allow for accurate determination of nucleosome positions before and during remodeling. Our findings suggest that these arrays can be efficiently chromatinized by salt gradient dialysis (SGD) and that remodeling can be performed as previously described. In our preliminary findings we validate that the optimizations to the DNA template have a potential to improve accuracy in determining the nucleosome position.
Taken together, in this thesis I document the technological advancements and novel, previously unobtainable, insights that characterize the S. cerevisiae chromatin landscape. Furthermore, these findings lay the foundation for future studies to apply an analogous approach on more complex, multicellular organisms and identify how chromatin regulates life.Eukaryotische Zellen verpacken und schützen ihre Erbinformationen mit Hilfe von Chromatin. Chromatin ist für diese Zellen überlebenswichtig. Nukleosome sind die Grundbausteine des Chromatins. Sie sind entlang der DNA wie Perlen auf einer Schnur positioniert. Die Nukleosome sind jedoch nicht etwa zufällig entlang des Genoms verstreut. In der Nähe der Startstelle für die Transkription befindet sich im Allgemeinen eine nukleosomenfreie Region (NFR). Diese wird gefolgt von vielen Nukleosomen, die in sehr regelmäßigen Abständen angeordnet sind. Wenn man dieses sog. Nukleosomen-‚Array‘ in verschiedenen Zellen kartiert, bemerkt man, dass es sich an erstaunlich ähnlichen Stellen befindet. Man spricht in dem Zusammenhang von einem phasierten ‚Array‘. Diese stereotype Organisation von Nukleosomen ermöglicht die Kontrolle der Genexpression und wird daher als ein wichtiges regulatorisches Element angesehen.
Einen großen Einfluss auf die Position von Nukleosomen haben ATP-abhängige Chromatin-‚Remodeling‘-Enzyme. Diese Enzyme können synergistisch oder antagonistisch zueinander wirken und übernehmen häufig redundante Rollen bei der Positionierung von Nukleosomen im Genom. Mutationen oder Deletionen dieser Enzyme können zu Instabilität des Genoms, zum Verlust der Regelmäßigkeit der Nukleosomen-‘Arrays‘ oder zu verminderter Phasierung führen, wodurch die Heterogenität in der Chromatinlandschaft erhöht wird. Neben ‚Remodelern‘ beeinflussen andere Faktoren wie die DNA-Sequenz und Allgemeine Regulatorische Faktoren (GRFs) die Nuklesomenlandschaft. Die Nukleosomenlandschaft kann zwischen verschiedenen Zellytpen oder in Abhängigkeit des Zellzyklus variieren. Aufgrund der Vielzahl unterschiedlicher Einflussfaktoren ist es aber schwierig, die Biogenese der Chromatinlandschaft zu analysieren.
In dieser Arbeit untersuchen wir die Biogenese und Heterogenität der Chromatinlandschaft in Saccharomyces cerevisiae. Zu diesem Zweck haben wir zunächst einen neuartigen Sequenzierungsansatz entwickelt, der es uns ermöglicht, die Lage von Nukleosomen auf einer einzelnen Chromatinfaser zu untersuchen. Wir zeigen, dass dieser Ansatz einzigartige Einblicke in die Nukleosomenorganisation der DNA bietet. Er ermöglicht es uns, die Heterogenität von Zelle zu Zelle zu bestimmen. Auch können wir feststellen, welche Enzymfaktoren die Regelmäßigkeit des ‚Arrays‘ hervorrufen. Wir beschreiben detailliert die Versuchsabläufe und Analysen, die zu einer optimalen Reproduzierbarkeit, Genauigkeit und hohem Durchsatz führen.
Zuerst wendeten wir unsere neuartige Technik an, um den rDNA-Locus zu untersuchen. Unsere Ergebnisse bestätigen eine zuvor beschriebene Dichotomie in der Chromatinlandschaft dieses Locus. Sie stimmen jedoch nicht mit dem vorgeschlagenen transkriptionell unabhängigen Charakter einzelner rDNA-Wiederholungseinheiten überein. Des Weiteren nutzten wir unsere Technik um die Heterogenität von Nukleosomen-‚Arrays‘ zu bestimmen. Hier fokussierten wir uns u.a. auf diejenigen Regionen im Genom, die mit traditionellen Techniken als unregelmäßig klassifiziert wurden. Wir stellten fest, dass trotz fehlender Phasierung fast alle dieser ‚Arrays‘ in Wildtypzellen eine hohe Regelmäßigkeit aufweisen. Zellen, denen ISWI- und CHD-Remodeler fehlen, besitzen hingegen ein tatsächlich unregelmäßige ‚Arrays‘, die nicht auf fehlende Phasierung zurückgeführt werden können. Des Weiteren untersuchten wir, inwiefern die Reduzierung der Nukleosomendichte die Arbeit von ‚Remodeling‘ Enzymen beeinflusst. Nach der ‚Klemmen‘ Theorien sollte eine Reduzierung der Dichte die Nukleosomenabstände nicht beeinflussen. Unsere Ergebnisse, gepaart mit Computermodellierungen, deuten aber auf einen nur schwachen Beitrag dieser molekularen ‚Klemmen‘ hin. Unsere Ergebnisse stützen hingegen frühere Ergebnisse unseres Labors, dass INO80 ein Remodeler ist, der einen regelmäßigen Abstand zwischen Nukleosomen einstellen kann. Mit Hilfe einer Variante unserer Technik können wir zwei verschiedene Hefestämme miteinander mischen und dadurch gleichzeitig analysieren. Vorläufige Ergebnisse zeigen, dass wir so globale Veränderungen der Nukleosomendichte höchst sensitiv detektieren können.
Im letzten Kapitel dieser Arbeit untersuchen wir unter Verwendung unseres Sequenzierungsansatzes, wie ISWI-Remodeler regelmäßige Abstände zwischen Nukelsomen in vitro einstellt. Frühere Ergebnisse aus unserem Labor haben gezeigt, dass die ‚Remodeling‘ Kinetik mit einem in vitro rekonstituierten Nukleosomen-‚Array‘ in Kombination mit Restriktionsendonukleasen untersucht werden kann. Diese Ergebnisse liefern jedoch keine Details zum Remodellierungsprozess und wie Nukleosomen auf einer einzelnen Faser bewegt werden. Zu diesem Zweck entwarfen wir zunächst eine neuartige DNA-Sequenz, die mehrere Widom 601-Nukleosompositionierungssequenzen enthält, die für unseren Sequenzierungsansatz optimiert wurden. Diese Optimierungen sollen eine genauere Bestimmung der Nukleosomenpositionen vor und während des ‚Remodelings‘ ermöglichen. Unsere Ergebnisse zeigen, dass diese ‚Arrays‘ durch Salzgradientendialyse (SGD) effizient chromatinisiert werden können und dass das ISWI diese ,Arrays’ als Substrat verwenden kann. In unseren vorläufigen Ergebnissen bestätigen wir, dass die Optimierungen der DNA Sequenz das Potenzial haben, die Genauigkeit bei der Bestimmung der Nukleosomenpositionen zu verbessern.
Insgesamt dokumentiere ich in dieser Arbeit die technologischen Fortschritte und neuartigen Einsichten in die Chromatinlandschaft von S. cerevisiae. Darüber hinaus legen diese Ergebnisse die Grundlage für zukünftige Studien, um mit Varianten unserer Technik Chromatin komplexerer, mehrzelliger Organismen zu untersuchen, in der Hoffnung, besser zu verstehen, wie Chromatin das Leben der Zelle reguliert
Conflict detection in software-defined networks
Get PDFThe SDN architecture facilitates the flexible deployment of network functions. While promoting innovation, this architecture induces yet a higher chance of conflicts compared to conventional networks. The detection of conflicts in SDN is the focus of this work.
Restrictions of the formal analytical approach drive our choice of an experimental approach, in which we determine a parameter space and a methodology to perform experiments. We have created a dataset covering a number of situations occurring in SDN. The investigation of the dataset yields a conflict taxonomy composed of various classes organized in three broad types: local, distributed and hidden conflicts. Interestingly, hidden conflicts caused by side-effects of control applications‘ behaviour are completely new.
We introduce the new concept of multi-property set, and the ·r (“dot r”) operator for the effective comparison of SDN rules. With these capable means, we present algorithms to detect conflicts and develop a conflict detection prototype. The evaluation of the prototype justifies the correctness and the realizability of our proposed concepts and methodologies for classifying as well as for detecting conflicts.
Altogether, our work establishes a foundation for further conflict handling efforts in SDN, e.g., conflict resolution and avoidance. In addition, we point out challenges to be explored.
Cuong Tran won the DAAD scholarship for his doctoral research at the Munich Network Management Team, Ludwig-Maximilians-Universität München, and achieved the degree in 2022. He loves to do research on policy conflicts in networked systems, IP multicast and alternatives, network security, and virtualized systems. Besides, teaching and sharing are also among his interests
Characterization of GAD-reaktive B cells and their B-cell receptor repertoire in the periphery and central nervous system
Get PDFAntibodies (abs) against the intracellular cytoplasmic protein glutamic acid decarboxylase 65 (GAD65) occur in different neurological disorders. Hereby, patients with anti-GAD65 ab associated autoimmune neurological disorders (GAD-AIND) exhibit high levels of anti-GAD65 abs in the serum as well as in the cerebrospinal fluid (CSF). However, the sites of immunological dysregulation and the triggers for the production of auto-abs against GAD65 are unclear. Furthermore, investigations on the pathogenic relevance of anti-GAD65 abs and their producing cells have yielded conflicting results. In consequence, the treatment of patients with GAD-AIND remains challenging. Therefore, investigating the functional properties of GAD-reactive B cells and anti-GAD65 abs could help to gain insights into the pathophysiology of GAD-AIND and assist the development of new treatment strategies.
To this end, we analyzed GAD-reactive B cells in peripheral blood of patients with GAD-AIND as well as bone marrow (bm) cells of one patient. As a next step, we generated monoclonal patient-derived abs from peripheral blood (PB) and CSF cells and characterized their reactivity to GAD in different assays. Moreover, we analyzed the auto-abs for sequence specificities, e.g., subclass distribution, and somatic hypermutations (SHMs).
In the first study, we could detect anti-GAD65 ab producing cells derived from GAD-reactive B cells in the peripheral blood (PB) of fifteen patients with GAD-AIND. Compared to our healthy control group consisting of nineteen donors, cells producing anti-GAD65 abs were highly elevated. Furthermore, we could identify bm plasma cells as an additional source of anti-GAD65 abs. The frequency of GAD-reactive B cells was comparable to B cells reactive for common recall antigens (ags) e.g. tetanus toxoid. In a second study, we included six patients with GAD-AIND and were able to generate 30 monoclonal abs (mabs) with 25 mabs derived from CSF cells and 5 mabs derived from PB cells. Mabs could be generated in patients with a short disease duration at the time of sampling. Thereby, we identified 10 GAD65-reactive mabs (CSF: 6/25; PB: 4/5) with enzyme-linked-immunosorbent-assay (ELISA). Furthermore, we could demonstrate that the intrathecal anti-GAD65 response is polyclonal and that the mabs had undergone affinity maturation when being compared to non-GAD65-reactive mabs. By reverting the SHMs of three GAD-reactive mabs to their germline sequences, we could show that affinity maturation is essential for the reactivity towards GAD65.
Taken together, our data characterize the B cell response in the CSF of patients with GAD-AIND, showing that the anti-GAD65 response is polyclonal and affinity maturation is necessary for the recognition of GAD65. Additionally, we could for the first time proof that anti-GAD65-ab-producing cells are present in the central nervous system. In addition, our data support the clinical observation that a B-cell depleting therapy might not be suitable for the treatment of long-standing GAD-AIND
Detektion von Imaging Biomarkern zur Charakterisierung und Prognoseabschätzung bei Patienten mit metastasiertem kolorektalen Karzinom
Get PDFIdentifikation genetischer Marker bei Kindern mit VACTERL-Assoziation
Get PDFThe VACTERL association is a rare malformation complex consisting of vertebral defects, anorectal malformation, cardiovascular defects, tracheoesophageal fistulae with esophageal atresia, renal malformation, and limb anomalies. As far as currently known, VACTERL is based on a multifactorial pathogenesis including genomic alterations.
The aim of this study was to improve our understanding of the genetic processes involved in the development of VACTERL. For this purpose, the genetic background was investigated with a focus on cardiovascular development, signalling pathways and cilia function. The study was designed as a genetic association study. Whole-exome sequencing (WES) followed by functional enrichment analysis was performed in 21 patients with VACTERL or VACTERL-like phenotype. To investigate the inheritance, the parents were examined by WES respectively Sanger sequencing.
In this study a genetic background for vascular disorders was identified in patients with VACTERL. This supports the theory, that congenital vascular defects may indicate other malformations in the spectrum of the VACTERL association. Additionally, this study indicates three more damage mechanisms for VACTERL. These are a general disruption of Shh- and Wnt signalling pathway by changes in signalling pathway activity, a structural cilia defect and a disruption of the ciliary signal transduction.
Our work unites these damage mechanisms in different, partly hereditary combinations and thus offers an explanation for the heterogeneity of the VACTERL association. Follow-up studies in cell and animal models should further address these combined damage mechanisms
