Transcriptional adaptation in caenorhabditis elegans

Transcriptional adaptation is a recently described phenomenon by which a mutation in one gene leads to the transcriptional modulation of related genes, termed adapting genes. At the molecular level, it has been proposed that the mutant mRNA, rather than the loss of protein function, activates this response. While several examples of transcriptional adaptation have been reported in zebrafish embryos and in mouse cell lines, it is not known whether this phenomenon is observed across metazoans. Here we report transcriptional adaptation in C. elegans, and find that this process requires factors involved in mutant mRNA decay, as in zebrafish and mouse. We further uncover a requirement for Argonaute proteins and Dicer, factors involved in small RNA maturation and transport into the nucleus. Altogether, these results provide evidence for transcriptional adaptation in C. elegans, a powerful model to further investigate underlying molecular mechanisms.publishedVersio

Establishment and application of genetic tools in the crustacean Parhyale hawaiensis

The main goal in the first part of this thesis was to develop a site specific recombination system in the arthropod crustacean Parhyale hawaiensis in order to exploit the recently developed transformation and gene trapping technology for functional comparative studies in arthropods. For that reason, I chose the PhiC31 integration system, which has been successfully used in Drosophila and in human cell lines. The PhiC31 bacteriophage inserts its genome into the host’s DNA by using specific recombination between two sites: attB (bacterial attachment point) and attP (phage attachment point).The recognition and recombination of these sites is catalysed by a site specific integrase, encoded by the phage’s genome. First, I created transgenic amphipods carrying the attP site and tested the possibility of introducing a heat inducible reporter gene through site specific recombination. After confirming that specific recombination occurs in Parhyale, I replaced the fluorescent marker gene of a gene trap. The conversion of a DsRed trap (distal) to a GFP expressing trap was highly efficient, suggesting that the integrase system could be used as a means of routinely converting gene traps into various genetic tools in Parhyale hawaiensis. The gene trapping and trap conversion constructs used in this study incorporate elements with proven activity in different species (3xP3, fluorescent proteins, PhiC31 integrase, splice acceptor sequence). Therefore, these same constructs could serve as a universal platform for gene trapping and trap conversion in diverse organisms. In the second part of the thesis, done in collaboration with the postdoctoral researcher Dr. Tasos Pavlopoulos from the University of Cambridge, we studied the role of the homeotic gene Ubx on appendage development and specialisation in crustaceans. The diversity of appendage morphology seen in crustaceans today, is the outcome of the evolution of an ancient homogenous trunk into specialised regions adapted for distinct functions. Various genetic mechanisms that could produce this diversity have been proposed, but functional evidence to test many of these hypotheses is still missing. The evolution of maxillipeds, which are thoracic limbs differentiated for feeding in crustaceans, represents one such example. Based on the correlation between the absence of the homeotic gene Ubx and the development of maxillipeds in several crustacean species, Averof and Patel proposed in 1997 that a shift of Ubx expression from anterior thoracic segments could serve as a mechanism for the specification of the maxilliped fate. The establishment of heat inducible gene expression in Parhyale hawaiensis gave us the opportunity to test this hypothesis directly in the amphipod by ectopically expressing Ubx in the first thoracic appendages, that normally develop into maxillipeds. Our results show that Ubx is sufficient to induce the transformation of maxillipeds into thoracic limbs, while complementary experiments of Ubx repression (from the laboratory of NH Patel) indicate its necessity to define thoracic identity. To explain the unexpected transformation of the maxillary appendages into maxillipeds by ectopic expression of Ubx, we studied the effect of Ubx on another homeotic gene Sex combs reduced (Scr), showing that this transformation was an indirect effect of the Ubx missexpression. Finally, this study allowed us to describe a large range of partial transformations of the maxillary appendages towards the thoracic fate, indicating that changes in the expression of a homeotic gene (like Ubx) can provide a great morphological diversity for natural selection to act upon.Στόχο της παρούσας διατριβής αποτέλεσε η ανάπτυξη ενός συστήματος ειδικού ανασυνδυασμού στο αρθρόποδο καρκινοειδές Parhyale hawaiensis με σκοπό την αξιοποίηση της τεχνολογίας μετασχηματισμού και γονιδιακής παγίδευσης, για την διεξαγωγή λειτουργικών συγκριτικών μελετών στα αρθρόποδα. Για το λόγο αυτό, επιλέχθηκε το σύστημα του φάγου φC31, που έχει χρησιμοποιηθεί με επιτυχία στη δροσόφιλα και σε ανθρώπινες κυτταρικές σειρές. Ο φάγος φC31 ενθέτει το γονιδίωμά του στο γένωμα του ξενιστή με την χρήση ειδικού ανασυνδυασμού ανάμεσα στις περιοχές attB (βακτηριακό σημείο πρόσδεσης) και attP (φαγικό σημείο πρόσδεσης). Την αναγνώριση των περιοχών αυτών και την κατάλυση της αντίδρασης ανασυνδυασμού, αναλαμβάνει μια ιντεγκράση που κωδικοποιείται από το γονιδίωμα του φάγου. Αφού δημιουργήθηκαν διαγονιδιακά άτομα που φέρουν την περιοχή attP στο γένωμα, ελέγχθηκε η δυνατότητα εισαγωγής με ειδικό ανασυνδυασμό ενός θερμοεπαγώμενου γονιδίου μάρτυρα. Μετά την επιβεβαίωση του ειδικού ανασυνδυασμού, επιχειρήθηκε η αντικατάσταση του γονιδίου μάρτυρα μιας γονιδιακής παγίδευσης. Η υψηλή συχνότητα μετατροπής μιας παγίδευσης (distal) από DsRed σε eGFP, ανοίγει τον δρόμο στην χρήση του συστήματος του φάγου φC31 στην αξιοποίηση των παγιδεύσεων που θα προκύψουν από τη σάρωση του γονιδιώματος του Parhyale hawaiensis. T Επιπλέον, η ενσωμάτωση εργαλείων με αποδεδειγμένη ενεργότητα σε διαφορετικά είδη (3xP3, φθορίζουσες πρωτεΐνες, ιντεγράση φC31, αλληλουχίες αποδοχής ματίσματος) καθιστούν την τελική κατασκευή έναν πιθανά οικουμενικό φορέα, για την εύκολη παγίδευση και κλωνοποίηση νέων γονιδίων και την διεξαγωγή λειτουργικών πειραμάτων σε νέους οργανισμούς. Στο δεύτερο μέρος της διατριβής, που έγινε σε συνεργασία με τον μεταδιδακτορικό ερευνητή Δρ. Τάσο Παυλόπουλο του πανεπιστημίου του Κέημπρητζ, παρουσιάζεται ο ρόλος του ομοιωτικού γονιδίου Ubx στην ανάπτυξη και εξέλιξη των άκρων στα καρκινοειδή. Η ποικιλομορφία που παρατηρούμε σημερα στην μορφολογία των άκρων στα καρκινοειδή, είναι προϊόν της εξέλιξής τους ώστε να ανταποκριθούν σε νέες ανάγκες και να καλύψουν καλύτερα παλαιότερες. Οι γενετικοί μηχανισμοί με τους οποίους μπορεί να παραχθεί αυτή η ποικιλομορφία ήταν μέχρι σήμερα άγνωστοι. Βασιζόμενοι στην συσχέτιση της απουσίας του ομοιωτικού γονιδίου Ubx με την ανάπτυξη των maxillipeds, που αποτελούν θωρακικά άκρα διαφοροποιημένα να επιτελούν λειτουργίες χειρισμού τροφής, οι Averof και Patel πρότειναν το 1997 την υποχώριση της έκφρασης του Ubx από τα πρώτα θωρακικά μεταμερίδια ως πιθανό μηχανισμό εμφάνισης αυτού του τύπου άκρου. Εκμεταλλευόμενοι τα νέα εργαλεία μετασχηματισμού και θερμοεπαγώμενης υπερέκφρασης, καθώς και την ευκολία γενετικού χειρισμού στο αμφίποδο Parhyale hawaiensis, θελήσαμε να ελέγξουμε αυτή την υπόθεση προσφέροντας εκτοπικά Ubx στο πρώτο θωρακικό μεταμερίδιο που αναπτύσσει maxillipeds. Τα αποτελέσματά μας δείχνουν πως το Ubx είναι ικανό να “επαναφέρει” στα maxillipeds τη μορφολογία θωρακικών άκρων, ενώ συμπληρωματικά πειράματα καταστολής του Ubx (από το εργαστήριο του NH Patel) υποδεικνύουν και την αναγκαιότητά του στον καθορισμό της “θωρακικής ταυτότητας”. Για να εξηγήσουμε το αναπάντεχο αποτέλεσμα της εμφάνισης maxillipeds από την εκτοπική έκφραση του Ubx σε κεφαλικά μεταμερίδια, μελετήσαμε την αλληλεπίδραση του Ubx με ένα άλλο ομοιωτικό γονίδιο (Scr), και δείξαμε πως ο φαινότυπος των maxillipeds μπορεί να προκύψει και με διαφορετικό μηχανισμό. Τέλος, ο μεγάλος αριθμός μετασχηματισμών, μας έδωσε την δυνατότητα να περιγράψουμε ένα ευρή φάσμα φαινοτύπων μετατροπής ενός κεφαλικού εξαρτήματος (maxilla) σε ένα βαδιστικό άκρο, υποδεικνύοντας την ποικιλομορφία που μπορούν να προσφέρουν για φυσική επιλογή, μικρές μόνο αλλαγές στην έκφραση ενός γονιδίου όπως το Ubx

Beyond Mendelian Inheritance: Genetic Buffering and Phenotype Variability

Understanding the way genes work amongst individuals and across generations to shape form and function is a common theme for many genetic studies. The recent advances in genetics, genome engineering and DNA sequencing reinforced the notion that genes are not the only players that determine a phenotype. Due to physiological or pathological fluctuations in gene expression, even genetically identical cells can behave and manifest different phenotypes under the same conditions. Here, we discuss mechanisms that can influence or even disrupt the axis between genotype and phenotype; the role of modifier genes, the general concept of genetic redundancy, genetic compensation, the recently described transcriptional adaptation, environmental stressors, and phenotypic plasticity. We furthermore highlight the usage of induced pluripotent stem cells (iPSCs), the generation of isogenic lines through genome engineering, and sequencing technologies can help extract new genetic and epigenetic mechanisms from what is hitherto considered ‘noise’.ISSN:2730-5848ISSN:2730-583

Beyond Mendelian Inheritance: Genetic Buffering and Phenotype Variability

Understanding the way genes work amongst individuals and across generations to shape form and function is a common theme for many genetic studies. The recent advances in genetics, genome engineering and DNA sequencing reinforced the notion that genes are not the only players that determine a phenotype. Due to physiological or pathological fluctuations in gene expression, even genetically identical cells can behave and manifest different phenotypes under the same conditions. Here, we discuss mechanisms that can influence or even disrupt the axis between genotype and phenotype; the role of modifier genes, the general concept of genetic redundancy, genetic compensation, the recently described transcriptional adaptation, environmental stressors, and phenotypic plasticity. We furthermore highlight the usage of induced pluripotent stem cells (iPSCs), the generation of isogenic lines through genome engineering, and sequencing technologies can help extract new genetic and epigenetic mechanisms from what is hitherto considered ‘noise’

Beyond Mendelian Inheritance: Genetic Buffering and Phenotype Variability

Understanding the way genes work amongst individuals and across generations to shape form and function is a common theme for many genetic studies. The recent advances in genetics, genome engineering and DNA sequencing reinforced the notion that genes are not the only players that determine a phenotype. Due to physiological or pathological fluctuations in gene expression, even genetically identical cells can behave and manifest different phenotypes under the same conditions. Here, we discuss mechanisms that can influence or even disrupt the axis between genotype and phenotype; the role of modifier genes, the general concept of genetic redundancy, genetic compensation, the recently described transcriptional adaptation, environmental stressors, and phenotypic plasticity. We furthermore highlight the usage of induced pluripotent stem cells (iPSCs), the generation of isogenic lines through genome engineering, and sequencing technologies can help extract new genetic and epigenetic mechanisms from what is hitherto considered 'noise'

Fast but not furious: A streamlined selection method for genome-edited cells

In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods

Fast but not furious: a streamlined selection method for genome-edited cells

In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods

PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants

Background The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents. Results We developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ Conclusion With PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.ISSN:1471-210

PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants

BACKGROUND The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents. RESULTS We developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ CONCLUSION: With PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications