Protein-protein interactions in human pluripotent stem cell-derived neural stem cells and their neuronal progeny

While most approaches in cell-based disease modeling are focused on the effects of defined mutations on the molecular or cellular phenotype, the assessment of underlying alterations in the interactomes of disease-relevant proteins has faced several technical challenges. First, experiments were typically conducted using overexpression paradigms resulting in unphysiologically high protein levels and thus promoting unspecific interactions. Second, such studies have been relying mostly on transformed cell lines, which enable mass production of transgenic cells but do not exhibit a tissue-specific proteomic environment. For that reason, the present study aimed at addressing these issues by bacterial artificial chromosome (BAC)-based expression of tagged proteins in pluripotent stem cell-derived long-term neuroepithelial like stem cells (lt-NES cells), a stable and robust cell population, which generates authentic human neurons with high fidelity. Tagged proteins were found to be expressed at endogenous levels, and fluorescence in situ hybridisation (FISH) analyses revealed an average integration rate of one copy per genome for the majority of cell lines analyzed. Correct compartmentalization and size of the tagged proteins could be confirmed by high-resolution confocal and live cell imaging as well as Western immunoblotting analysis, respectively. Employing this approach, multiple cell lines were generated harboring tagged proteins associated with human developmental disorders, cancer and neurodegeneration. Representatives of these groups include Proliferating Cell Nuclear Antigen (PCNA), Aurora Kinase A (AURKA), Cyclin-Dependent Kinase 2-Associated Protein 1 (CDK2AP1), Set Domain-Containing Protein 1B (SETD1B), RuvB-Like 2 (RUVBL2), the Methyl CpG Binding Protein 2 (MECP2) and the Alzheimer’s disease-associated proteins Nicastrin (NCSTN) and Valosin-Containing Protein (VCP). Using a label-free, quantitative affinity purification-mass spectrometry approach, numerous novel interaction partner candidates of these proteins were identified. Direct comparison of protein-specific interactomes of proliferating lt-NES cells and their neuronal progeny further revealed changes in the composition of several chromatin-remodeling complexes, suggesting that this system is sufficiently sensitive and specific to identify the dynamic differential recruitment of individual proteins as a response to developmental switches. In a proof-of-concept study, the approach of BAC-mediated expression of tagged proteins with a subsequent analysis of interacting proteins was successfully transferred to induced pluripotent stem cell (iPS)-derived lt-NES cells in order to enable PPI analyses in the context of complex diseases in following studies. Finally, an adeno-associated virus based approach for epitope tagging of endogenous genes in iPS-derived lt-NES cells from a patient suffering from Machado-Joseph disease allowed the generation of cell pools exhibiting both the diseased and healthy isoform of N-terminal FLAG-tagged Ataxin-3. The present work demonstrates a successful establishment of two different methods for protein tagging in somatic cell populations that subsequently can be employed for a multitude of analytical techniques including fluorescent microscopic visualization of protein localization, dynamics of protein recruitment or the detection of PPI

Determinants of the initiation of meiotic recombination

Spo11, zuerst in Saccharomyces cerevisiae beschrieben, ist ein eukaryotisches Homolog der archealen DNS Topoisomerase VIA Untereinheit und ist für die Bildung von DNS Doppelstrangbrüchen (DSB) während der Meiose notwendig. Die DNS wird bevorzugt an bestimmten Stellen im Genom, welche meiotische Rekombinations Hotspots genannt werden, geschnitten. Nach dem DNS Schnitt wird Spo11 mit einem kovalent gebundenen Oligonukleotid von der Bruchstelle gelöst. Diese kurze einzelsträngige DNS Sequenz entspricht genau dem Ort des Doppelstrangbruches. Das Arabidopsis thaliana Genom kodiert, anders als bei Säugern und Hefe, wo es nur ein Spo11 gibt, für drei Spo11 Homologe, AtSPO11-1, AtSPO11-2 und AtSPO11-3. Nur AtSPO11-1 und AtSPO11-2 sind für die Meiose notwendig, während AtSPO11-3 an der somatischen Endoreduplikation beteiligt ist. Bei S. cerevisae sind zusätzlich zu Spo11 zumindest 9 andere Proteine an der DSB Bildung beteiligt. Bei Arabidopsis sind bisher nur die drei Proteine AtPRD1, AtPRD2 und AtPRD3, als essentiell für die SPO11 abhängige DSB Bildung, identifiziert worden. Das Hauptthema dieser Arbeit war die Entwicklung eines Protokolls, um genomweit DSB Stellen in Arabidopsis thaliana zu identifizieren. Zuerst wurde ein Deep-Sequencing basierendes Protokoll unter Zuhilfenahme eines Modellsubstrates, einem HRP Protein gekoppelt an ein 30 Nukleotide langes Oligonukleotid, entwickelt und optimiert. Nach der erfolgreichen Optimierung der einzelnen Reaktionsschritte und der Sequenzierung wurden Saccharomyces cerevisiae und Schizosaccharomyces pombe verwendet, um die Methode zu testen und die Resultate mit denen von bereits publizierten DSB Karten zu vergleichen. Die Resultate für beide Hefen waren sehr gut reproduzierbar und es wurde eine gute Übereinstimmung mit den genomischen Hotspot Karten, die bereits mit anderen Methoden produziert wurden, gefunden. Um diese Methode auf Arabidopsis thaliana zu übertragen, wurde eine transgene Linie, welche eine funktionelle, getaggte Version von AtSPO11-1 (AtSPO11-1/18xmyc) exprimiert, generiert. Das Protein wurde erfolgreich aus Pflanzenmaterial, welches meiotische Zellen enthält, präzipitiert und für das oben genannte Protokoll verwendet. Die ersten Deep-Sequencing Durchgänge, welche mit Arabidopsis Material durchgeführt wurden, brachten 3,5 Millionen und 2,2 Millionen Sequenzen, welche zum Arabidopsis Genom passen. Ein weiteres Ziel dieser Arbeit war die gezielte Stimulation von meiotischer Rekombination in A. thaliana. Eine spezifische DNS Bindedomäne wurde an den C-Terminus von AtSPO11-1 fusioniert (AtSPO11-1/Gal4) und es wurden Pflanzen hergestellt, die korrespondierende Cis-Elemente (5xUAS) tragen. Nach dem Kreuzen der Pflanzen mit den vorher genannten Features wird erwartet, dass AtSPO11-1/Gal4 an die 5xUAS Stellen bindet, um in weiterer Folge die Bildung von meiotischen Doppelstrangbrüchen zu stimulieren. Weiters wurde versucht, Interaktionspartner von AtSPO11-1 und AtSPO11-2 zu finden. Für diesen Zweck wurden verschiedene Strategien, wie Massenspektrometrie, direkte Yeast two-hybrid Interaktion und Co-Immunopräzipitation verwendet.Spo11, first described in Saccharomyces cerevisiae, is a eukaryotic homolog of the archeal DNA topoisomerase VIA subunit and is needed for the formation of DNA double strand breaks (DSBs) during meiosis. DNA is preferentially cleaved at certain sites in the genome, called meiotic recombination hotspots. After DNA cleavage, the Spo11 protein is released from the cleavage site with a short DNA oligonucleotide covalently attached. This short ssDNA sequence corresponds to the exact meiotic DSB cleavage site. The Arabidopsis thaliana genome encodes, unlike the ones of mammals and yeast, where only one Spo11 is present, three Spo11 homologs, AtSPO11-1, AtSPO11-2 and AtSPO11-3. Only AtSPO11-1 and AtSPO11-2 are essential for meiosis, whereas AtSPO11-3 is needed for somatic endoreduplication. In S. cerevisiae, in addition to Spo11, at least 9 other proteins are essential for meiotic DSB formation. In Arabidopsis only three further proteins, AtPRD1, AtPRD2 and AtPRD3, have been identified to be essential for SPO11 mediated meiotic DSB formation. The main topic of this thesis was the development of a protocol for the identification and detailed analysis of meiotic DSB sites in the model plant Arabidopsis thaliana. First, a deep-sequencing based protocol was developed and optimized using an HRP protein, conjugated to a 30mer oligonucleotide, as a model substrate. After successful optimization of the individual reactions steps and the deep-sequencing pipeline, Saccharomyces cerevisiae and Schizosaccharomyces pombe were used as model organisms to verify the method and to compare the results to known DSB maps. Results for both yeasts were very well reproducible and found very good overlap with genome-wide hotspot maps generated earlier by other methods. To implement the method in the higher eukaryote Arabidopsis, a transgenic line expressing a functional, tagged version of AtSPO11-1 (AtSPO11-1/18xmyc) was generated. AtSPO11-1/18xmyc could be successful immuno-precipitated from plant material containing meiotic cells and be used in the protocol mentioned above. The first two deep-sequencing runs, using material from Arabidopsis have been performed, yielding 3.5 million and 2.2 million sequence reads that mapped to the Arabidopsis genome. An additional aim of this thesis was the targeted stimulation of meiotic recombination in A. thaliana. A specific DNA binding domain was fused to the C-terminus of AtSPO11-1 (AtSPO11-1/Gal4) and plant lines carrying corresponding cis-elements (5xUAS) were generated. After crossing plants with aforementioned features it is anticipated that the tagged AtSPO11-1 will be guided to the cis-elements and stimulate formation of meiotic DSBs. Furthermore it was aimed to find AtSPO11-1 and AtSPO11-2 interacting proteins. For this purpose, different approaches like mass spectrometry, direct yeast two-hybrid interaction and co-immuno-precipitation were used

Part I: Studies on the Drosophila selector gene apterous and compartment boundary formation

This PhD Thesis is divided into two different, separate parts. In the first part, I focus on the transcriptional regulation of the Drosophila selector gene apterous (ap). During animal development, selector gene activity is known to be important for the subdivision of cell populations into distinct functional units, called compartments. ap is essential for the subdivision into a dorsal and ventral compartment of the wing imaginal disc. This compartmentalization is a prerequisite for proper wing development. While the function of ap as a dorsal selector gene has been studied extensively, the regulation of its expression during wing development is poorly understood. In the presented studies, the transcriptional regulation of ap was analyzed by classical means and extended by novel approaches, which allowed direct manipulation of the endogenous locus. By combining all those approaches, we identified three separable cis-regulatory elements that work in synergy to regulate the expression of ap during wing imaginal disc development and gained insight into the general patterning of the wing disc and the de novo formation of a compartment boundary. In the second part, I focus on the development and application of a novel class of protein binders, called nanobodies. Protein-protein interactions are key to almost all biological processes. So far, protein functions in vivo have been mostly studied by genetic manipulations. However, to describe and understand protein functions in their respective native environment, it is very important and necessary to manipulate proteins directly in vivo. Towards this end, the discovery and development of a new class of protein binders (nanobodies) was essential. Nanobodies are protein binders based on single-domain antibody scaffolds. Conveniently, randomized nanobody libraries have been engineered that hypothetically allow the isolation of nanobodies against any protein of interest. As the field of protein binders is still very young, we wanted to explore the possibility to generate novel specific nanobodies. Using phage display, I did isolate new specific nanobodies. Importantly, we demonstrated that these new nanobodies work intracellularly in cell culture and in vivo

Modelling the structural, functional and phenotypic consequences of protein coding mutations

Proteins are integral to all cellular processes and underpin the function of all extant organisms, meaning variants impacting them are a primary cause of phenotypic variation. Protein coding variants are a key area of study in biology, with relevance from structural and molecular biology to population genetics. They are also medically important, impacting inherited genetic diseases, cancer and response to pathogens. Recent advances in highthroughput experimental techniques have opened the door to many new approaches in biology, and protein variants are no exception. Deep mutational scanning experiments exhaustively measure the fitness of variants in a protein, which gives us more experimentally validated mutational consequence measurements than ever before. Such advances, together with ever larger sequence and structure databases, have created an opportunity to apply large scale analyses to coding variation, studying the effect on protein structure, function and phenotype. In this thesis I perform three large scale variant analyses. First, I use the consequences of variation to learn about protein structure and function. I compile a dataset from 28 deep mutational scanning studies, covering 6291 positions in 30 proteins, and use the consequences of mutation at each position to define a mutational landscape. I show rich biophysical relationships in this landscape and identify functionally distinct positional subtypes of each amino acid. In the second analysis, I explore genotype to phenotype prediction using a dataset of 1011 S. cerevisiae strains, with genotypes, transcriptomics, proteomics and measured phenotypes, and comprehensive gene deletions in four strains. I show knowledge-based models of mutational consequences and pathway function can be used to associate genes with phenotypes and predict growth phenotypes across 34 growth conditions. However, genetic background is found to have a large effect on variant consequences, to such an extent that the same deletion can be highly significant in one strain and have no effect in another. Finally, I analyse computational variant effect prediction, benchmarking current predictors using deep mutational scanning data. I then develop a new end-to-end deep convolutional neural network predictor that predicts consequences directly from sequence and structure and show it improves on current methods. Together these projects advance our knowledge of protein coding variation and enhance our capacity to link variation to impacts on structure, function and phenotype

New views on the Drosophila transcriptome

Drosophila is a valuable experimental organism can be used as a reverse genetics model. Drosophila Malpighian (renal) tubules are important epithelial tissue in which to study transport mechanisms. RNA-seq has been chosen to investigate Drosophila Malpighian (renal) tubules to identify novel genes following a three- way comparison between three popular transcriptome profiling methods. Two types of novel gene have been found in Drosophila tubules, coding genes and noncoding genes. Reverse genetics has been applied to identify novel coding gene function in Drosophila tubules. Three-way analysis of Drosophila expression microarrays, Drosophila tiling micrarrays and Drosophila RNA-seq reveal that most gene expression levels are well correlated between the three technologies. Drosophila expression microarrays and RNA-seq are correlated better than the correlation between Drosophila tiling microarrays and RNA-seq. Drosophila expression arrays and Drosophila tiling arrays all suffered from cross-hybridization, miss target detection and hybridization background noise, and also have low dynamic range for detecting lowly and highly expressed genes. Drosophila tiling microarrays also have a high false-positive detection rate, which may lead to overestimate the transcriptional activities of the genome. RNA-seq has overcome the drawbacks of microarrays and become the leading technology for genome sequencing, transcriptome profiling, novel gene discovery, and novel alternative splicing discovery with wide dynamic range. However, Drosophila expression microarrays and tiling microarrays still remain useful. Three-prime expression microarrays offer a means to measure the differential three-prime end processing, and tiling microarrays can be used for novel gene discovery. In this sense, the three technologies complement each other. Poly(A) selected RNA-seq has been used as a discovery tool for searching novel genes in Drosophila Malpighian tubules in this thesis. A TopHat and Cufflinks pipeline has been used as an analytical pipeline for novel gene discovery and differential gene expression analysis between Drosophila tubules and whole flies in order to find the tubule-enriched genes. Reverse genetics has been applied to Drosophila to achieve a gene knockdown and overexpression by using the unique Gal4/UAS system to achieve the novel gene knockdown or overexpression in specific tissue and cell types. Novel coding gene CG43968 has been discovered. The location of this gene has been confirmed in tubule main segments, principle cell cytoplasm or apical membrane. The function of this gene has been identified as involvement in tubule secretion, which may relate to calcium transport. Reverse genetics has been confirmed as particularly important for the functional study of novel genes

Systems approaches to study root architecture dynamics

The plant root system is essential for providing anchorage to the soil, supplying minerals and water, and synthesizing metabolites. It is a dynamic organ modulated by external cues such as environmental signals, water and nutrients availability, salinity and others. Lateral roots (LRs) are initiated from the primary root post-embryonically, after which they progress through discrete developmental stages which can be independently controlled, providing a high level of plasticity during root system formation. Within this review, main contributions are presented, from the classical forward genetic screens to the more recent high-throughput approaches, combined with computer model predictions, dissecting how LRs and thereby root system architecture is established and developed