Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). "20-July-2006." Includes bibliographical references. Also available via the World Wide Web.

Στο πλαίσιο της παρούσης διατριβής, αξιοποιήθηκαν οι αλληλουχίες γνωστών γονιδιωμάτων, αλλά και γονιδιωμάτων που αποκρυπτογραφήθηκαν πρόσφατα, για να μελετηθεί η εξελικτική ιστορία τριών λειτουργικά σημαντικών πρωτεϊνικών οικογενειών: (α) των φυτικών DNA μεθυλομεταφορασών και (β) των ευκαρυωτικών RNA μεθυλομεταφορασών, οι οποίες είναι ένζυμα που επιφέρουν μεθυλίωση στις νουκλεοτιδικές αλληλουχίες, καθώς και (γ) των πεπτιδασών συγγενών της καλλικρεΐνης (KLKs), οι οποίες είναι γνωστές σερινοπρωτεϊνάσες με δράση τύπου θρυψίνης ή χυμοθρυψίνης. Οι εξελικτικές σχέσεις των χαρακτηρισμένων και καινοφανών πρωτεϊνών των τριών οικογενειών διερευνήθηκαν με κατασκευή φυλογενετικών δένδρων. Επίσης, αναλύθηκε η δευτεροταγής και τριτοταγής δομή των ομόλογων πρωτεϊνών, η δομή των γονιδίων που κωδικοποιούν τις πρωτεΐνες αυτές, και κατασκευάσθηκαν διαγνωστικά πρωτεϊνικά μοτίβα από τις αλληλουχίες των τριών οικογενειών ενζύμων. Τα αποτελέσματα των αναλύσεων οδήγησαν στην εξαγωγή σημαντικών συμπερασμάτων σχετικά με την πιθανή βιολογική λειτουργία των καινοφανών πρωτεϊνών. Συγκεκριμένα, ομόλογες φυτικές DNA μεθυλομεταφοράσες και καινοφανείς ευκαρυωτικές RNA μεθυλομεταφοράσες ταυτοποιήθηκαν σε δημόσιες βάσεις δεδομένων. Λεπτομερής φυλογενετική ανάλυση οδήγησε στην ταυτοποίηση των τεσσάρων ήδη γνωστών οικογενειών φυτικών DNA μεθυλομεταφορασών και μιας καινοφανούς υποοικογένειας (Pavlopoulou and Kossida, 2007). Επίσης, ταυτοποιήθηκαν πέντε υποοικογένειες ευκαρυωτικών RNA μεθυλομεταφορασών. Πέραν των τριών ήδη γνωστών υποοικογενειών (NOP2, NCL1 και YNL022C), ταυτοποιήθηκε μια καινοφανής υποοικογένεια (RCMT9), και μια υποοικογένεια, FMU, η οποία εθεωρείτο ότι απαντάται αποκλειστικά σε προκαρυωτικούς οργανισμούς (Pavlopoulou and Kossida, 2009). Επιπλέον, κατασκευάσθηκαν πρωτεϊνικά αποτυπώματα της οικογένειας (και των επιμέρους υποοικογενειών) των RNA μεθυλομεταφορασών, τα οποία καταχωρήθηκαν στη δευτερογενή πρωτεϊνική βάση δεδομένων PRINTS (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). Στα πλαίσια της διατριβής, αναπτύχθηκε το υπολογιστικό πρόγραμμα RCMTHMM, με σκοπό τη διάκριση/ταυτοποίηση των ευκαρυωτικών RNA μεθυλομεταφορασών, το οποίο διατέθηκε για δημόσια χρήση στη διεύθυνση URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM. Σημαντική συνεισφορά της παρούσας μελέτης είναι η αναδιατύπωση της εξελικτικής ιστορίας των καλλικρεϊνών (Pavlopoulou et al., 2010). Οι καλλικρεΐνες είναι σημαντικά πρωτεολυτικά ένζυμα που δρουν ατομικά ή σε πρωτεολυτικούς καταρράκτες και ρυθμίζουν σημαντικές φυσιολογικές λειτουργίες, ενώ η απορρυθμισμένη δράση τους έχει συνδεθεί με σοβαρές ασθένειες (καρδιοαγγειακές, νευροεκφυλιστικές, φλεγμονώδεις, δερματικές, διάφορους τύπους καρκίνου). Για την απομόνωση νέων καλλικρεϊνών αξιοποιήθηκε το γεγονός ότι τα KLK γονίδια συνεντοπίζονται στο ίδιο χρωμόσωμα υπό μορφή μη διακοπτόμενης συστάδας γονιδίων. Σε προηγούμενες μελέτες είχε προταθεί ότι οι καλλικρεΐνες απαντώνται μόνον στα θηλαστικά και ότι εμφανίσθηκαν πριν από περίπου 150 εκατομμύρια χρόνια. Στην παρούσα μελέτη, ομόλογες καινοφανείς ακολουθίες καλλικρεϊνών ανιχνεύθηκαν in silico στα γονιδιώματα διάφορων οργανισμών. Ορθόλογα των καλλικρεϊνών ταυτοποιήθηκαν για πρώτη φορά στα ερπετά, στα πτηνά και στα αμφίβια, υποδεικνύοντας την εξελικτική καταγωγή των καλλικρεϊνών πριν από 330 εκατομμύρια χρόνια. Επιπροσθέτως, πέραν των 15 γνωστών KLKs (KLΚ1-15), ταυτοποιήθηκαν τρία καινοφανή μέλη (ορφανές Klks) και με σύγκριση των προβλεπόμενων δομών δείχθηκε ότι τα δομικά χαρακτηριστικά που σχετίζονται με την καταλυτική δράση είναι συντηρημένα στις καινοφανείς πρωτεϊνικές αλληλουχίες KLK. Σημειωτέον, δείχθηκε ότι στα γονιδιώματα όλων των υπό εξέταση οργανισμών, τα ορθόλογα γονίδια KLK χαρτογραφούνται στην ίδια χρωμοσωμική περιοχή, διευθετημένα εν σειρά με τον ίδιο προσανατολισμό και χωρίς παρεμβολή μη καλλικρεϊνικών γονιδίωνΠροτείναμε ότι η οικογένεια των καλλικρεϊνών προέκυψε από μια σειρά γονιδιακών διπλασιασμών και μεταλλάξεων, και ότι οι καλλικρεΐνες έχουν συνεξελιχθεί με τα ειδικά υποστρώματά τους (Pavlopoulou et al., 2010). / In the present thesis, the availability of an increasing number of complete or almost complete genomes, including those that were completed recently, enabled the study of the evolutionary history of three functionally important protein families: (a) the plant DNA methyltransferases and (b) the eukaryotic RNA methyltransferases, which are enzymes that catalyze the transfer of a methyl group to nucleotide sequences, as well as (c) the kallikrein-related peptidases or KLKs, which are trypsin- or chymotrypsin-like serine proteases. The evolutionary relationships of the already known and the novel proteins of the three families that were identified here were investigated using phylogenetic trees. Moreover, the secondary and tertiary structures of the homologous proteins were analyzed, as well as the structure of the protein-encoding genes, and diagnostic protein motifs were constructed based on the sequences of the three enzyme families. Our results led to suggestions pertaining to the biological function of the identified novel proteins. In particular, homologous plant DNA methyltransferases and novel eukaryotic RNA methyltransferases were identified in publicly accessible sequence databases. Detailed phylogenetic analysis of plant DNA methyltransferases identified four already known families and a novel subfamily in addition (Pavlopoulou and Kossida, 2007). Moreover, five distinct eukaryotic RNA methyltransferase subfamilies were identified; apart from the three already known subfamilies (NOP2, NCL1 and YNL022C), one novel subfamily (RCMT9) and the FMU which hitherto was considered to exist exclusively in prokaryotes were also identified (Pavlopoulou and Kossida, 2009). Furthermore, protein fingerprints were constructed from the generic family of RNA methyltransferases (and the individual subfamilies), which were deposited in the PRINTS database (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). We developed the computational program RCMTHMM, in order to discriminate/identify eukaryotic RNA methyltransferases from other proteins. The RCMTHMM program has been made publicly available in the URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM. Finally, the evolutionary history of KLKs was reconstructed. Kallikreins are important proteolytic enzymes which are involved in proteolytic cascade pathways and their dysregulated expression has been associated with major human pathologies (cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, skin diseases, different cancer types). The prominent feature of the kallikrein family is that it consists of tandemly and uninterruptedly arrayed genes on a single locus at human chromosome 19q13.3-13.4. This unique feature was used in order to identify novel KLKs and KLK-like genes/proteins. Previous studies on the evolution of kallikreins were restricted to mammals and the emergence of the kallikrein genes was suggested approximately 150 million years ago. In the present study, homologous novel kallikrein protein sequences were detected in silico in the genomes of various species. For the first time, novel KLK orthologues were identified in reptiles, aves and amphibia, which allowed us to trace the evolutionary origin of kallikreins 330 million years ago. In addition, apart from the 15 already known KLK genes (KLK1-15), three novel members were identified (orphan Klks). All the defining structural features which are related to the catalytic activity of KLKs were found to be conserved in the novel KLK protein sequences. Of particular interest, the synteny of the KLK-encoding genes was analyzed and it was shown that these genes are co-localized in contiguous, uninterrupted clusters maintaining the same orientation in all species under investigation. We suggest that a series of gene duplication and mutation events gave rise to the family of KLK enzymes and KLKs have co-evolved with their specific substrates (Pavlopoulou et al., 2010).

L'auteur n'a pas fourni de résumé en français / L'auteur n'a pas fourni de résumé en anglais

Philosophiae Doctor - PhD (Biotechnology) / The 'Bon Rouge' pear cultivar was developed from a bud mutation on a 'Bon Chretien' pear tree. The latter is characterised by green fruit skin and leaves, while 'Bon Rouge' is characterised by red leaves and red fruit skin as a result of the production of anthocyanin and other pigments. Branch forming buds on 'Bon Rouge' trees often revert to the parent phenotype producing green leaves and fruit skin. The occurrence of both phenotypes on the same tree presents a unique model to study gene expression associated with anthocyanin production in a similar genetic background under the same set of environmental condition.

African horse sickness virus (AHSV) affects equine populations around the world. It is the cause of a high rate of morbidity and associated large economic losses in affected regions. The virus is a segmented double stranded RNA virus and a member of Orbivirus genus in the Reoviridae family. The prototype member of the orbiviruses is bluetongue virus (STY) and other members include Chuzan virus and St. Croix River virus. These viruses are all characterized by a genome of ten dsRNA segments that encode at least ten different proteins. Three of the minor core proteins are found within the core of BTV. These are all associated with the RNA transcription complex and the enzymatic activities with which they are associated include an RNA polymerase (VP1), an RNA capping enzyme (VP4) and an RNA helicase (VP6). Genes homologous to the BTV genes that encode these proteins are found in all members of the Orbivirus genus. The aim of this thesis is to characterize VP4 of AHSV, the capping enzyme candidate, and to compare it to other orbivirus capping enzymes. Possible functional motifs and regions of importance within the orbivirus capping enzymes will be identified. The gene will also be expressed and used to perform assays to characterize the different enzymatic activities of VP4. The VP4 cDNA of AHSV serotype 3 was cloned and sequenced. From the full-length verified nucleotide sequence an open reading frame was identified and used to predict the amino acid sequence. These were compared to other orbivirus species including STY, Chuzan virus and St. Croix River virus. These alignments identified a number of highly conserved regions, consisting of four or more amino acids conserved between all the sequences analyzed. A fibronectin type 3-like motif, containing 12 conserved amino acids, was identified which could be responsible for protein binding. This motif contains 12 conserved amino acids making it a good candidate for a functional motif. Conservation does not, however, always predict regions of importance. In BTV a lysine-containing motif was identified to be responsible for GMP binding. This region is not conserved between the different viruses. AHSV has a motif containing a lysine residue similar to the motif identified in rotavirus and reovirus. Two other motifs described in BTV were also not conserved in the other viruses. One of them, a leucine zipper, was shown to dimerize BTV VP4. Phylogenetically, AHSV and Chuzan virus are the most closely related while BTV is more distant and St. Croix River virus forms a distinct out-group when the different VP4 sequences are compared. AHSV-3 VP4 was expressed as a histidine-tagged protein in the baculovirus expression system. Not unexpectedly, the protein was found to be insoluble, similar to BTV VP4 produced by means of the same system. However, whereas BTV VP4 could be solubilized by the addition of salt the AHSV VP4 remained insoluble at high salt concentrations. Several adjustments were made. Cells were lysed in a high salt buffer, the pH of the buffers was adjusted and sucrose cushions were used but none of the methods was found to improve the yield of soluble VP4 significantly. However, the pellet containing VP4 was relatively empty of contaminating protein and, therefore, a number of enzymatic assays were performed with the pellet. Assays for inorganic phosphatase and nucleotide phosphatase were performed. Strikingly, both assays indicated the presence of active phosphatases in the WT and VP4 pellets. Also, an assay was performed for guanylyltransferase activity but no activity was observed for this assay. The sequence data therefore points to VP4 as the probable capping enzyme although it may have a different structural complex. The failure to produce a reliable source of soluble purified AHSV VP4 made it impossible to provide evidence to confirm the associated enzymatic activities. / Dissertation (MSc(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

No description available.

Les mécanismes épigénétiques jouent un rôle essentiel dans la régulation de l’expression des gènes. L’enzyme du complexe répresseur Polycomb II EZH2, capable de triméthyler la lysine 27 de l’histone H3 (H3K27me3) est impliquée dans la régulation de nombreux processus normaux, tels que le développement et la différenciation cellulaire. Les plasmocytes jouent un rôle majeur dans la réponse immunitaire humorale. La différenciation des lymphocytes B en plasmocytes (PCD) est finement régulée par un réseau de facteurs de transcription impliqué de l’induction et le maintien de l’identité de ces deux types cellulaires. Par ailleurs, peu de mécanismes épigénétiques ont été décrits dans la PCD. En utilisant un modèle in vitro de PCD développé dans notre laboratoire, nous avons mis en évidence une surexpression d’EZH2 dans le stade préplasmablaste (prePB) de la PCD. Grâce à l’analyse globale des séquences d’ADN associées à EZH2 et H3K27me3 dans ce type cellulaire, nous avons montré que l’enzyme était capable de réprimer l’expression de gènes impliqués dans différentes fonctions des lymphocytes B et des plasmocytes. EZH2 est également capable de se fixer sur le promoteur de gènes actifs dans les prePB, impliqués dans la régulation de la prolifération de ces cellules. En outre, nous avons montré que l’inhibition chimique d’EZH2 dans notre modèle résultent en une dérégulation transcriptionnelle associée à une accélération du processus de différenciation. Nos résultats suggèrent qu’EZH2 est impliqué dans le maintien de l’état transitoire, immature et prolifératif des prePBs via la régulation de gènes, dépendante et indépendante de H3K27me3, favorisant l’amplification des cellules à défaut de leur différenciation en plasmocytes. Des anomalies de séquence ou de l’expression d’EZH2 ont été mis en évidence de nombreux cancers hématologiques et solides. Le myélome multiple (MM) est une hémopathie caractérisée par l’accumulation de plasmocytes tumoraux dans la moelle osseuse. Nos travaux ont notamment permis d’identifier une surexpression des membres du complexe Polycomb Repressive Complex 2 (PRC2) dans les cellules de MM, en association avec leur prolifération. Afin de comprendre le rôle de PRC2 dans le MM, nous avons utilisé un inhibiteur de l’activité méthyltransférase d’EZH2 (EPZ-6438). L’effet de l’inhibiteur d’EZH2 sur la prolifération et la survie des cellules de MM est très hétérogène. En effet, les cellules sensibles présentent un arrêt du cycle cellulaire et entrent en apoptose. De manière intéressante, la résistance des cellules de MM à l’inhibiteur d’EZH2 pourrait être médiée induite par la méthylation des promoteurs des gènes cibles de PRC2. Nous avons ainsi établi un score (EZ-score), basé sur l’expression des gènes, permettant d’identifier des patients de mauvais pronostic pouvant bénéficier d’un traitement avec un inhibiteur d’EZH2. Nous avons également mis en évidence un effet synergique de EPZ-6438 et du Lenalidomide, un agent immuno-modulateurs utilisé en traitement conventionnelle du MM. Cette inhibition de la croissance cellulaire est notamment due à l’induction de l’expression de facteurs de transcription, spécifiques des lymphocytes B, et des suppresseurs de tumeurs en association avec la répression de l’expression de MYC. Aussi, un prétraitement avec l’inhibiteur d’EZH2 permet de surmonter la résistance des cellules tumorales au Lenalidomide. Ces données suggèrent que le ciblage de PRC2 pourrait avoir un intérêt thérapeutique chez les patients caractérisés par un mauvais pronostic et un fort EZ-score. Ainsi, l’inhibiteur d’EZH2 pourrait également permettre de resensibiliser les patients aux chimiothérapies basées sur des agents immuno-modulateurs. / Epigenetic mechanisms play an essential role in gene expression regulation. EZH2, the catalytic sub-unit of PRC2, is able to trimethylate the lysine 27 of histone H3 (H3K27me3) and is involved in the regulation of numerous normal processes, such as development and cell differentiation. Plasma cells (PCs) play a major role in the defense of the host organism against pathogens, by producing antigen-specific antibodies. B cell differentiation into PC is mediated by a fine-tuned regulatory network of cell specific transcription factors involved in B and plasma cell identity. Although numerous key actors involved in plasma cell differentiation (PCD) have been described, most of the epigenetic mechanisms associated with this process are yet to be unveiled. Using an in vitro model of PCD developed in our laboratory, we showed that EZH2 is upregulated in the preplasmablast stage (prePB) of the PCD. By analyzing DNA sequences associated with EZH2 and H3K27me3 in this cell type, we highlighted that EZH2 is recruited to and represses through H3K27me3 a subset of genes involved in B cell and plasma cell identity. Interestingly, in prePBs and PBs, EZH2 was also found to be recruited to H3K27me3-free promoters of transcriptionally active genes known to regulate cell proliferation and DNA repair. Inhibition of EZH2 catalytic activity resulted in B to PC transcriptional changes associated with PC maturation induction together with higher immunoglobulin secretion. Altogether, our data suggests that EZH2 is involved in the maintenance of prePBs/PBs transitory immature proliferative state through H3K27me3-dependent and independent gene regulation supporting their amplification. Moreover, while EZH2 overexpression was previously shown to inhibit PCD in mice, this study highlights for the first time that EZH2 inhibition can accelerate normal human PCD by prematurely inducing a plasma cell transcriptional program.EZH2 mutations or abnormal expression were shown to be involved in numerous hematological malignancies and solid tumors. Multiple Myeloma (MM) is a malignant plasma cell disease with a poor survival, characterized by the accumulation of myeloma cells (MMCs) within the bone marrow. We identified a significant upregulation of the Polycomb Repressive Complex 2 (PRC2) core genes in MM cells in association with proliferation. We used EPZ-6438, a specific small molecule inhibitor of EZH2 methyltransferase activity, to evaluate its effects on MM cells phenotype and gene expression profile. PRC2 targeting results in cell growth inhibition due to cell cycle arrest and apoptosis together with Polycomb, DNA methylation, TP53 and RB1 target genes induction. EZH2 inhibitor induced toxicity was heterogeneous in human myeloma cell lines and primary MM cells from patients. Interestingly, we found that MM cell resistance to EZH2 inhibitor could be mediated by DNA methylation of PRC2 target genes. We established a gene expression-based EZ-score allowing to identify poor prognosis patients that could benefit from EZH2 inhibitor treatment. We also demonstrated a synergistic effect of EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment, through the activation of B cell transcription factors and tumor suppressor gene expression in concert with MYC repression. Moreover, EPZ-6438 pre-treatment was able to overcome MM cells resistance to lenalidomide. These data suggest that PRC2 targeting could have a therapeutic interest in MM patients characterized by high-risk EZ-score values, reactivating B cell transcription factors and tumor suppressor genes. EZH2 inhibitor could also re-sensitize MM patients to chemotherapies based on immunomodulatory agents.

DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as D. melanogaster and C. elegans. In this thesis we report on a comprehensive study of the pres- ence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. We observe that loss of individual DNMTs independently occured multiple times across ecdysozoan phyla. In several cases, this resulted in a loss of DNA methylation. In vertebrates, however, there is no single species known which lost DNA methylation. Actually, DNA methylation was greatly expanded after the 1R/2R whole genome duplication (WGD) and became a genome-wide phe- nomena. In our study of vertebrates we are not looking for losses of DNA methyltransferases and DNA methylation but are rather interested in the gain of additional DNA methyltransferase genes. In vertebrates there were a number of WGD. Most vertebrates only underwent two WGD but in the teleost lineage a third round of WGD occured and in some groups, e.g. Salmoniformes and some Cypriniformes even a forth WGD occured. The Carp-specific WGD (4R) is one of the most recent vertebrate WGD and is estimated to have occured 12.4 mya. We performed the most comprehen- sive analysis of the evolution of DNA methyltransferases after vertebrate whole-genome duplications (WGD) so far. We were able to show that the conservation of duplicated DNMT3 genes in Salmoniformes is more diverse than previously believed. We were also able to identify DNA methyltrans- ferases in Cypriniformes which have, due to their recent WGD, quite com- plex genomes. Our results show that the patterns of retained and lost DNA methyltransferases after a forth round of WGD differ between Cypriniformes and Salmoniformes. We also proposed a new nomenclature for teleost DNMT genes which correctly represents the orthology of DNMT genes for all teleost species. Next to these purely computational projects we collaborated with the Aluru lab to investigate the effects of different disturbances on zebrafish DNA methylation. One disturbance is the inactivation of DNMT3aa and DNMT3ab as single knockouts as well as a double knockout. This was the first double knockout of DNMT genes in zebrafish which was ever generated. It allows us to study the subfunctionalization of the two DNMT3a genes their effect on genome-wide DNA methylation. Given our results we hypothesize that DNMT3aa and DNMT3ab can compensate for each other to a high de- gree. DNMT3a genes have likely been subfuntionalized but their loss can be compensated by DNMT3b genes. This compensation by DNMT3b genes works well enough that no notable phenotype can be observed in double knockout zebrafish but a difference is notable on the epigenome level. The second disturbance we studied is the exposure of zebrafish to the toxic chemi- cal PCB126. We detected a moderate level of DNA methylation changes and a much larger effect on gene expression. Similar to previous reports we find little correlation between DNA methylation and gene expression changes. Therefore, while PCB126 exposure has a negative effect on DNA methyla- tion it is likely that other gene regulatory mechanisms play a role as well, possibly even a greater one. How do genes evolve and how are genes regulated are two of the main questions of modern molecular biology. In this thesis we have tried to shed more light on both questions. we have broadly expanded the phylogenetic range of species with a manually curated set of DNA methyltransferases. We have done this for ecdysozoan species which have lost all DNA methylating enzymes as well as for teleost fish which acquired more than ten copies of the, originally, two genes. We were also able to generate new insight into the subfunctionalization of the DNA methylation machinery in zebrafish and how it reacts to environmental effects.:1 Introduction 1.1 Biological introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2 Detecting DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Evolution of DNA methylation across Ecdysozoa 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3 Evolution of DNA methyltransferases after vertebrate whole genome duplications 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 The effect of DNMT3aa and DNMT3ab knockout on DNA methyla- tion in zebrafish 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish exposed to Pentachlorobiphenyl 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 6 Conclusions 6.1 Evolution of DNA methylation across Ecdysozoa . . . . . . . . . . . . . 95 6.2 Evolution of DNA methyltransferases after vertebrate whole genome duplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 6.3 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish (Danio rerio) exposed to Pentachlorobiphenyl (PCB 126). . . 107 6.4 Knockout of DNMT3aa and DNMT3ab in zebrafish (Danio rerio) . . . . . . 108 Bibliography 119

In multicellular organisms, all the cells are genetically identical but turn genes on or off at the right time to promote differentiation into specific cell types. The regulation of higher-order chromatin structure is essential for genome-wide reprogramming and for tissue-specific patterns of gene expression. The complexity of the genome is regulated by epigenetic mechanisms, which act at the level of DNA, histones, and nucleosomes. Epigenetic machinery is involved in many biological processes, including genomic imprinting, X-chromosome inactivation, heterochromatin formation, and transcriptional regulation, as well as DNA damage repair. In this review, we summarize the recent understanding of DNA methylation, cytosine derivatives, active and passive demethylation pathways as well as histone variants. DNA methylation is one of the well-characterized epigenetic signaling tools. Cytosine methylation of promoter regions usually represses transcription but methylation in the gene body may have a positive correlation with gene expression. The attachment of a methyl group to cytosine residue in the DNA sequence is catalyzed by enzymes of the DNA methyltransferase family. Recent studies have shown that the Ten-Eleven translocation family enzymes are involved in stepwise oxidation of 5-methylcytosine, creating new cytosine derivatives including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Additionally, histone variants into nucleosomes create another strategy to regulate the structure and function of chromatin. The replacement of canonical histones with specialized histone variants regulates accessibility of DNA, and thus may affect multiple biological processes, such as replication, transcription, DNA repair, and play a role in various disorders such as cancer.

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