Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-12-13T18:53:37Z No. of bitstreams: 2 Tese - Natalia do Carmo Silva - 2017.pdf: 2078936 bytes, checksum: a9911e8e1a6da63e31e4fb86a1dbf785 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-14T10:19:33Z (GMT) No. of bitstreams: 2 Tese - Natalia do Carmo Silva - 2017.pdf: 2078936 bytes, checksum: a9911e8e1a6da63e31e4fb86a1dbf785 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-14T10:19:33Z (GMT). No. of bitstreams: 2 Tese - Natalia do Carmo Silva - 2017.pdf: 2078936 bytes, checksum: a9911e8e1a6da63e31e4fb86a1dbf785 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this study was to evaluate the addition of antioxidants in different cryopreservation diluents on bovine semen quality after thawing and the in vitro production of embryos. First, the effects of adding different concentrations of glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM) in egg yolk semen cryopreservation diluents (Bovimix®) and soy lecithin (Andromed®) were evaluated. The results showed that the addition of the antioxidant glutathione did not improve the maintenance of sperm characteristics of bovine semen. Melatonin had a negative effect on the spermatic parameters evaluated. The diluent Andromed® obtained better parameters of spermatic kinetics when compared with the diluent Bovimix®. However, in relation to the membrane integrity analyzes of the Bovimix® diluent showed better performance in the maintenance of these parameters. The effect of the cryopreservation diluents and the addition of glutathione in the vitro production of bovine embryos (PIVE) was then evaluated. The rate of cleavage, blastocyst rate and hatching were evaluated eight and nine days after IVF, as well as the correlation between sperm characteristics and PIVE. The diluent Andromed® provided a better cleavage rate, with no effect of the addition of glutathione in it. However, the addition of 2.5 mM glutathione in the Bovimix® diluent improved the cleavage rate. There was a significant correlation of high and low magnitude between some sperm characteristics with the rate of cleavage. It was concluded that glutathione did not improve sperm viability. Melatonin worsened the maintenance of sperm characteristics. Andromed® diluent was more efficient in the in vitro production of bovine embryos with no glutathione effect observed in this diluent. The addition of 2.5 mM glutathione in the egg yolk Bovimix® diluent gave a higher cleavage rate. In vitro evaluation of semen quality is not the best method to predict the fertilizer potential of the same in PIVE, because only significant low to moderate magnitude correlations were observed between some parameters of semen quality and fertilization rate. / Objetivou-se avaliar a adição de antioxidantes em diferentes diluentes de criopreservação sobre a qualidade do sêmen bovino pós-descongelação e a produção in vitro de embriões. Primeiramente, avaliou-se os efeitos da adição de diferentes concentrações de glutationa (1,5 e 2,5 mM) e melatonina (0,5 e 1,0 mM) em diluentes de criopreservação de sêmen à base de gema de ovo (Bovimix®) e lecitina de soja (Andromed®). Os resultados demonstraram que a adição do antioxidante glutationa não melhorou a manutenção das características espermáticas do sêmen bovino. A melatonina reduziu a qualidade das características espermáticas avaliadas. O diluente Andromed® obteve melhores variáveis de cinética espermática quando comparado com o diluente Bovimix®. No entanto, com relação às análises de integridade de membrana o diluente Bovimix® demonstrou melhor desempenho na manutenção dessas variáveis. Posteriormente, avaliou-se o efeito dos diluentes de criopreservação e a adição de glutationa sobre a produção in vitro de embriões bovinos (PIVE). Avaliou-se a taxa de clivagem, taxa de blastocisto e eclosão oito e nove dias após a fertilização (FIV), assim como a correlação entre as características espermáticas e as taxas de desenvolvimento embrionário. O diluente Andromed® proporcionou melhor taxa de clivagem, sem a necessidade da adição de glutationa no mesmo. Entretanto, a adição de 2,5 mM de glutationa no diluente Bovimix® melhorou a taxa de clivagem. Houve correlação significativa de alta e baixa magnitude entre algumas características espermáticas com a taxa de clivagem. Conclui-se que a glutationa não melhorou a viabilidade espermática. A melatonina reduziu a manutenção das características espermáticas. O diluente Andromed® foi mais eficiente na produção in vitro de embriões bovinos não sendo observado efeito da glutationa neste diluente. A adição de glutationa na concentração de 2,5 mM no diluente Bovimix®, à base de gema de ovo, proporcionou maior taxa de clivagem. A avaliação in vitro da qualidade do sêmen não é o melhor método para predizer o potencial fertilizante do mesmo na PIVE, pois só foram observadas correlações significativas de baixa a moderada magnitude entre algumas características de qualidade do sêmen e a taxa de fertilização.

Le cortisol est reconnu pour être une hormone clé dans le maintien de la balance hydrominérale en eau douce et dans l'adaptation à l'eau de mer, chez de nombreux téléostéens juvéniles. Cependant, son rôle au cours du développement embryonnaire est encore mal connu, notamment son implication dans le développement des cellules spécialisées dans le transport ionique, les ionocytes. L'objectif de ma thèse a été de déterminer l'implication du cortisol lors de la mise en place du lignage des ionocytes de la peau chez l'embryon de médaka (Orysias latipes) puis d'étudier les conséquences d'une élévation du cortisol embryonnaire sur les capacités osmorégulatrices des larves lors d’un transfert dans une eau pauvre en ions ou en eau de mer. Dans un premier temps, une attention particulière a été portée à la dynamique d'apparition des ionocytes de la peau du sac vitellin des embryons. Ces derniers apparaissent en deux vagues successives avec une cinétique propre. Nous avons alors proposé un modèle de développement des ionocytes pour chacune de ces vagues. Grâce à cette première étude, nous avons ensuite montré que du cortisol exogène ne modifie pas le taux de prolifération et/ou de différenciation des ionocytes épidermiques mais accélère leur différenciation. De plus, nous avons identifié un des récepteurs aux glucocorticoïdes (GR2) comme régulateur de l’ontogenèse des ionocytes, très probablement grâce à ces transcrits maternels. Enfin, nous avons montré que les larves de médaka sont capables de réguler très rapidement leurs contenus en ions Na+ et Cl- après de chocs hypo- et hyper-osmotiques. En revanche, la capacité des larves à réguler les contenus en Ca2+ est plus limitée lors d’un choc hypo-osmotique. Un doute important sur l’efficacité du traitement cortisol lors de cette dernière partie ne nous permet pas de mettre en lien le rôle du cortisol dans l’ontogenèse des ionocytes avec la fonction d’osmorégulation de ces derniers à l’éclosion. Ces travaux ont donc permis d’établir les bases de l’ontogenèse des ionocytes embryonnaires ainsi que de l’osmorégulation des larves chez le médaka pour la caractérisation du rôle du cortisol et de ses récepteurs. De façon similaire, ce modèle pourra être utilisé comme support pour l’identification et la caractérisation de nouveaux régulateurs. / Cortisol is a key hormone regulating in teleost fish water and ionic homeostasis in freshwater and seawater and in acclimation during salinity changes. However, its role during embryonic stages is still poorly known, especially its involvement in the development of ionic transport specialized cell, namely the ionocytes. The aim of my thesis was to determine cortisol involvement in epidermal ionocyte lineage establishment in medaka (Orysias latipes) embryos and to study consequences of cortisol elevation in medaka embryos on larval osmoregulatory abilities during transfer from freshwater to ion-poor environment or to seawater transfer. In a first part, we studied the dynamic of ionocyte appearance in yolk-sac epithelium of embryos. Ionocytes appear in two distinct waves with their own kinetic. This allowed us to propose a model of ionocyte development for each wave. In the continuity of this first part, we have showed that exogenous cortisol doesn’t modify the proliferation and/or differentiation rate of epidermal ionocytes but rather accelerate their differentiation. In addition, we have identified GR2, one of glucocorticoid receptors, as the main regulator of ionocyte ontogenesis, most likely through its maternal transcripts. Finally, we have showed that medaka larvae are able to quickly regulate their Na+ and Cl- ion contents after hypo- or hyper-osmotic challenges. In contrast, larvae ability to regulate Ca2+ ion contents is more limited during hypo-osmotic challenge. A doubt on the effectiveness of the cortisol treatment, in this last part, prevent us to understand the relationship between cortisol role in ionocyte ontogenesis and its osmoregulatory functions after hatching. These studies have established in medaka the basis of embryonic ionocyte ontogenesis and larval osmoregulation in order to clarify the role of cortisol and its receptors. Similarly, this fish model could be used as a support for identification and characterization of new regulators of the osmoregulation function.

<div><p>Zika virus (ZIKV) is a mosquito-borne pathogen that stayed unnoticed for over half a century. Only after the 2015-16 Brazilian outbreak did the severity of the infectious outcome, particularly the Congenital Zika Syndrome, become apparent. ZIKV is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly, ventriculomegaly, retinopathy, and sensorineural hearing loss. Though the pandemic is now under control in the Latin American countries, several tropical countries could still be at risk of widespread infection. This warrants a better understanding of the congenital Zika syndrome; this project attempts to contribute towards this goal.</p><p><br></p><p>Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in non-uniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8, expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell autonomous changes in brain patterning.</p><p><br></p></div><p>Sensorineural hearing loss is a relatively understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. ZIKV pathogenesis was explored in the developing inner ear using the accessible chicken embryo model system. One goal was to assess the spatiotemporal susceptibility of otic epithelial-derived structures to ZIKV infectivity. Direct injections of the inner ear or the inner ear primordium were performed <i>in ovo</i>with subsequent harvests at 2 to 8 days-post-infection. The degree of infection in sensory/prosensory organs was evaluated histologically to determine the susceptibility of one auditory and five vestibular organs. ZIKV infection of the sensory as well as non-sensory epithelia was observed at most stages of analysis, with no apparent preference for one over the other. The lagena, the ventral most tip of the chicken inner ear, and the endolymphatic sac/duct were least frequently infected. In this report, two novel findings in sequela of ZIKV infection are presented: the vestibular labyrinth can present with stalled canal morphogenesis, and the auditory ganglion can be severely shrunken, perhaps due to an increased cell death upon early ZIKV infection of the inner ear.</p><p><br></p><p>Additional methods of peripheral infection in the chicken embryos were tested to examine ZIKV transmission to the central nervous system: E3 blood vessel, E4 limb bud, and E10 chorioallantoic membrane infections. Although none of these methods resulted in a histologically significant infection of the developing brain 3 to 6 days-post-infection, evidence of ZIKV genome replication and viremia was detected in several tissue types.<br></p>

Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood. In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment. These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z.:Frontmatter II Acknowledgements VII Thesis Summary (English) IX Thesis Summary (German) X Table of Contents XIV List of Figures XVI List of Tables XVII List of Abbreviations XXIII 1 Introduction 1 1.1 Transcription regulation 2 1.1.1 Promoter elements - genetic information that guides transcription initiation 2 1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3 1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4 1.2 Chromatin 4 1.2.1 Histone variants 7 1.2.2 Posttranslational histone modifications 7 1.2.3 Histone Lysine methylation 8 1.2.4 H3K4me3 in embryonic development 10 1.3 Establishment and removal of H3K4me3 10 1.3.1 Set1 homologs - Set1a and Set1b 11 1.3.2 Trithorax homologs - Mll1 and Mll2 11 1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13 1.3.4 COMPASS complex proteins 13 1.3.5 H3K4me3 removal 14 1.4 Transcription activation in embryos 14 1.4.1 Zebrafish early embryonic development 15 1.4.2 H3K4me3 during early zebrafish development 17 1.5 Thesis aim 17 2 Materials and Methods 19 2.1 Materials 19 2.2 Methods 36 2.2.1 Zebrafish husbandry and care 36 2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36 2.2.3 Generation of plasmids for mRNA production 38 2.2.4 Microinjection 39 2.2.5 Germline transplantation 39 2.2.6 Western Blot Assays 40 2.2.7 RNA extraction and quantification assays 41 2.2.8 Chromatin immunoprecipitation (ChIP) 43 2.3 Bioinformatics Analyses 46 2.3.1 Quality control, alignment and peak calling 46 2.3.2 Lambda normalization 46 2.3.3 Differential ChIP enrichment analysis 47 2.3.4 Data integration 47 2.3.5 Gene classification 48 3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49 3.1 Generation and phenotypic description of histone methyl-transferase mutants 49 3.1.1 HMT TALEN mutagenesis workflow 49 3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52 3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54 3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56 3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60 3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62 3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67 3.5 Conclusion 70 4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71 4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71 4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73 4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75 4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76 4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76 4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77 4.5 H3K4me3 levels at gene promoters are reduced upon introduction of methylation-defective Histone H3 79 4.6 Early transcription activation is not altered upon K4M overexpression 88 4.7 Conclusion 92 5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93 5.1 H3K4me3 levels increase over developmental time at all gene classes 93 5.2 H3K4me3 is gained at CpG-rich elements 98 5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100 5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101 5.5 Maternally provided genes are enriched for H2A.z and CpG content 103 5.6 Conclusion 104 6 Discussion 105 6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106 6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107 6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109 6.4 H3K4me3 potentially gains importance during later developmental stages 111 6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112 6.6 Conclusion 115 Appendix 117 Bibliography 139 Authorship Declaration 159 / Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt. In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen. Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen.:Frontmatter II Acknowledgements VII Thesis Summary (English) IX Thesis Summary (German) X Table of Contents XIV List of Figures XVI List of Tables XVII List of Abbreviations XXIII 1 Introduction 1 1.1 Transcription regulation 2 1.1.1 Promoter elements - genetic information that guides transcription initiation 2 1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3 1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4 1.2 Chromatin 4 1.2.1 Histone variants 7 1.2.2 Posttranslational histone modifications 7 1.2.3 Histone Lysine methylation 8 1.2.4 H3K4me3 in embryonic development 10 1.3 Establishment and removal of H3K4me3 10 1.3.1 Set1 homologs - Set1a and Set1b 11 1.3.2 Trithorax homologs - Mll1 and Mll2 11 1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13 1.3.4 COMPASS complex proteins 13 1.3.5 H3K4me3 removal 14 1.4 Transcription activation in embryos 14 1.4.1 Zebrafish early embryonic development 15 1.4.2 H3K4me3 during early zebrafish development 17 1.5 Thesis aim 17 2 Materials and Methods 19 2.1 Materials 19 2.2 Methods 36 2.2.1 Zebrafish husbandry and care 36 2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36 2.2.3 Generation of plasmids for mRNA production 38 2.2.4 Microinjection 39 2.2.5 Germline transplantation 39 2.2.6 Western Blot Assays 40 2.2.7 RNA extraction and quantification assays 41 2.2.8 Chromatin immunoprecipitation (ChIP) 43 2.3 Bioinformatics Analyses 46 2.3.1 Quality control, alignment and peak calling 46 2.3.2 Lambda normalization 46 2.3.3 Differential ChIP enrichment analysis 47 2.3.4 Data integration 47 2.3.5 Gene classification 48 3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49 3.1 Generation and phenotypic description of histone methyl-transferase mutants 49 3.1.1 HMT TALEN mutagenesis workflow 49 3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52 3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54 3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56 3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60 3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62 3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67 3.5 Conclusion 70 4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71 4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71 4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73 4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75 4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76 4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76 4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77 4.5 H3K4me3 levels at gene promoters are reduced upon introduction of methylation-defective Histone H3 79 4.6 Early transcription activation is not altered upon K4M overexpression 88 4.7 Conclusion 92 5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93 5.1 H3K4me3 levels increase over developmental time at all gene classes 93 5.2 H3K4me3 is gained at CpG-rich elements 98 5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100 5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101 5.5 Maternally provided genes are enriched for H2A.z and CpG content 103 5.6 Conclusion 104 6 Discussion 105 6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106 6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107 6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109 6.4 H3K4me3 potentially gains importance during later developmental stages 111 6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112 6.6 Conclusion 115 Appendix 117 Bibliography 139 Authorship Declaration 159

Since their emergence in the early 1990s, neonicotinoid use has increased exponentially to make them the world's most prevalent insecticides. Although there is considerable research concerning the lethality of neonicotinoids, their sub-lethal and developmental effects are still being explored, especially with regards to non-mammalian species. The goal of this research was to investigate the effects of the neonicotinoid imidacloprid on the morphological and physiological development of northern bobwhite quail (Colinus virginianus). Bobwhite eggs (n = 650) were injected with imidacloprid concentrations of 0 (sham), 10, 50, 100 and 150 grams per kilogram of egg mass, which was administered at day 0 (pre-incubation), 3, 6, 9, or 12 of growth. Embryos were dissected on day 19 when they were weighed, staged, and examined for any overt structural deformities. Embryonic heart, liver, lungs and kidneys were also weighed and preserved for future use. Treated embryos exhibited increased frequency of severely deformed beaks and legs, as well as larger hearts and smaller lungs at the higher dosing concentrations. Some impacts are more pronounced in specific dosing periods, implying that there may be critical windows of development when embryos are highly susceptible to neonicotinoid exposure. This investigation suggests that imidacloprid could play a significant role in chick survival and declining quail populations in treated regions of the country.

Chromatin is a dynamic structure, and chromatin remodeling enzymes regulate chromatin structure to control gene expression and proper lineage specification. Tip60-p400 is a multi-subunit chromatin remodeling complex containing two biochemical activities: the Tip60 subunit is a lysine acetyltransferase (KAT) that targets histones and non-histone proteins, and p400 catalyzes ATP-dependent incorporation of histone variant H2AZ into chromatin. Both of these chromatin modifications have been widely studied with respect to gene expression, DNA damage repair, and apoptosis. Ablation of these catalytic subunits causes defects in normal embryonic development, ESC self-renewal, and gene expression. My goal has been to understand the multiple independent functions of Tip60-p400 acetyltransferase in ESC maintenance and embryonic development. I showed that Tip60 KAT function is dispensable for gene expression, chromatin accessibility, and ESC self-renewal, which is different from Tip60 knockdown phenotype. Interestingly, KAT deficient mutants exhibited defect in differentiation towards mesoderm and endoderm lineages. Consistent with this defect, I also observed gastrulation defect in mice lacking Tip60 KAT activity. Together, these data demonstrate that Tip60 KAT dependent function is only required during later stages of embryonic development, and it is dispensable for ESC self-renewal and pre-implantation development. Tip60 KAT contains four isoforms generated from alternative splicing, whose individual functions are poorly characterized. In the second part of this thesis, I investigated the developmental role of one of the isoforms of Tip60, called Tip55. Unlike Tip60 knockout mice, which lack all the isoforms and causes pre-implantation lethality, I found that ablation of Tip55 results in post-implantation lethality. I further found that loss of Tip55 causes defects in heart, and neural tube development, demonstrating the essential function of Tip55 isoform for organogenesis during embryonic development. Together, these studies have provided new insight into the functions of Tip60-p400 and the mechanisms by which this complex regulates gene expression, ESC pluripotency, and embryonic development. Furthermore, these studies set the stage for future work to identify how the catalytic and non-catalytic functions are directed to perform distinct regulatory functions, as well as how each Tip60 isoform individually contributes to formation of the mammalian body plan.

Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control. Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease. The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain. We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys. We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia. Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease. In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.

No description available.

Regulatorische Landschaften von Genen steuern das präzise transkriptionelle Programm, das für die embryonale Entwicklung notwendig ist. Transkriptionsfaktoren (TFs) interagieren dabei mit regulatorischen Elementen (REs), um die Genexpression zu kontrollieren. Zur Untersuchung der zugrundeliegenden Mechanismen konzentriere ich mich auf das Xist-Gen, den Hauptregulator der X-Chromosom-Inaktivierung (XCI) in Säugetieren. In der Embryonalentwicklung wird Xist monoallelisch in weiblichen Zellen aktiviert, woraufhin die Xist-RNA das X-Chromosom überzieht und dessen Inaktivierung einleitet. Dadurch wird die erhöhte Dosis X-chromosomaler Gene in weiblichen Zellen kompensiert. Um ein umfassendes Verständnis der Xist-Regulatoren zu erhalten, nutze ich CRISPR-Screens, um REs und TFs in weiblichen embryonalen Stammzellen zu untersuchen. Dabei identifiziere ich ein neues nicht-kodierendes Gen namens Xert. Daruberhinaus stelle ich fest, dass promotor-nahe REs auf die Anzahl der X-Chromosomen reagieren, während distale REs unbeeinflusst bleiben. Durch meine TF-Screens entdecke ich zwei Gruppen von Aktivatoren: Die frühe Gruppe, darunter der X-chromosomale Faktor ZIC3, zeigt in weiblichen Zellen erhöhte Expression, was darauf hindeutet, dass sie die Xist-Expression auf weibliche Zellen beschränken. Die späte Gruppe, einschließlich OTX2, agiert geschlechtsunabhängig und stellt nach der initialen Xist-Aktivierung ein hohes Transkriptionslevel sicher. Mit weiteren CRISPR-Screens verknüpfe ich TFs mit REs und zeige, dass frühe Aktivatoren promotor-nahe REs beeinflussen, während späte Aktivatoren distale REs stärker regulieren. Diese Arbeit liefert eine systemische Perspektive des trans- und cis-regulatorischen Netzwerks, das die Xist-Aktivierung während der Differenzierung koordiniert und die Beschränkung auf weibliche Zellen gewährleistet. / The regulatory landscapes of developmental genes orchestrate precise and coordinated transcriptional programs required for embryonic development. During this process, transcription factors (TFs) interact with regulatory elements (RE) to finely tune gene expression. To study the regulatory principles acting in this context, I focus on the Xist gene, the master regulator of X-chromosome inactivation (XCI) in mammals. During early development, Xist is upregulated in a monoallelic fashion specifically in females. The Xist RNA then coats the X chromosome in cis, resulting in its inactivation. In this manner, female cells compensate for their increased X-chromosomal dosage in comparison to males. To obtain a complete understanding of Xist regulation, I first perform two CRISPR screens targeting REs and TFs during the early differentiation of female mouse embryonic stem cells. I identify Xist-controlling REs within the locus, unveiling a novel non-coding gene Xert. I further demonstrate the sensitivity of promoter-proximal REs to X-dosage, contrasted by the behavior of distal REs. In the TF screen, I detect two sets of activators which undergo transient upregulation at the onset of XCI. The early group of activators, including the X-linked TF ZIC3, exhibits higher expression levels in XX cells, indicating a role in restricting Xist expression to females. The late group of activators, including the master regulator of the epiblast OTX2, drives high transcript levels following Xist activation. Subsequently, I use a series of CRISPR screens targeting individual reporter constructs to map TF-RE wiring at the locus. I find that the early activators primarily act on the XX-dependent proximal REs. Contrary, the late activators interact with the sex-independent distal REs. With this study, I provide a systems level perspective of the trans- and cis-regulatory network that links Xist activation to early differentiation and ensures female-specificity.

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