1,733 research outputs found

    The Role of the HUSH complex and LINE-1 elements in the Regulation of Type 1 Interferon

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    Although Transposable Elements (TEs) have been a reliable source of genetic variation throughout evolution (Rishishwar et al., 2018), host genomes have simultaneously coevolved with TEs to employ a variety of strategies to regulate their aberrant activity. In fact, mounting evidence indicates the deleterious consequences of dysregulated TE expression in a number of human diseases including autoimmune and inflammatory disorders (M. K. Crow, 2010), monogenic diseases (Cordaux & Batzer, 2009; Nakamura et al., 2015) and various cancers (Helman et al., 2014; Lee et al., 2012). The goal of this project is to explore how the human genome continues to adapt to the ongoing evolutionary arms race between TEs and the innate immune defence system and to uncover novel epigenetic pathways that help suppress their aberrant activity and characterise how these pathways regulate innate immune genes. The Human Silencing Hub (HUSH) is an epigenetic silencing complex that is necessary for the repression of LINE-1 elements (Fukuda et al., 2018; N. Liu et al., 2018; Robbez-Masson et al., 2018). Here, we reveal the depletion of the HUSH complex component, MPP8, in human cell lines and primary fibroblasts leads to the induction of interferon-stimulated genes (ISGs) through JAK/STAT signalling and demonstrate that this effect is mainly attributable to MDA5 and RIG-I-mediated sensing of double-stranded RNAs (dsRNAs). This response coincides with the upregulation of primate-conserved LINE-1 elements, as well as increased expression of a subset of full-length hominid-specific LINE-1s that produce sense and antisense transcribed RNA products, which may form dsRNAs. Furthermore, we show that LINE-1 shRNAs could abrogate the HUSH-dependent response, while overexpression of an engineered full-length LINE-1 construct activates interferon signalling in somatic cells. Finally, we provide some insights into the physiological regulation of HUSH and HUSH-regulated LINE-1s during the normal immune response in primary human fibroblasts. Taken together, our results suggest that endogenous LINE-1s drive physiological and autoinflammatory responses through dsRNA sensing and gene-regulatory roles that are ultimately under HUSH control. Our work thus serves to highlight HUSH/MPP8 as a potential drug target for future cancer immunotherapies, where MPP8 inactivation may be harnessed to drive type 1 IFNs and anti-tumour immunity

    Dissecting structural and biochemical features of DNA methyltransferase 1

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    DNA methylation is an epigenetic modification found in every branch of life. An essential enzyme for the maintenance of DNA methylation patterns in mammals is DNA methyltransferase 1 (DNMT1). Its recruitment is regulated through its large N-terminus, which contains six annotated domains. Although most of these have been assigned a function, we are still lacking a holistic understanding of the enzyme's spatio-temporal regulation. Interestingly, a large segment of the N-terminus is devoid of any known domain and appears to be disordered in its sequence. Over the past years, such disordered sequences have increasingly gained attention, due to their role in forming biomolecular condensates through liquid-liquid phase separation (LLPS). These liquid compartments offer specific environmental conditions distinct from the surrounding that can enhance protein recruitment and function. In this work, we explore a potential role for the intrinsically disordered domain (IDR) in the recruitment of DNMT1. Taking an evolutionary approach, we uncover that structural features of the region that are key for IDR function are highly conserved. Moreover, we find conserved biochemical signatures compatible with a role in LLPS. Using a reconstitution assay and an opto-genetic approach in cells, we for the first time show that the DNMT1 IDR is capable of undergoing LLPS in vitro and in vivo. In addition, we define a novel region of interest (ROI) of about 120 amino acids in the IDR that appears to have been inserted in the ancestor of eutherian mammals. Although the ROI has a distinct biochemical signature, we find no effect on the LLPS behavior of the IDR. Therefore, we discuss other potential roles of the ROI related to DNA methylation, for example, imprinting. Finally, we lay the foundation for investigating a biological function of the IDR and establish a system for screening DNMT1 mutant phenotypes in mouse embryonic stem cells. Swift depletion of the endogenous protein is enabled by degron-mediated degradation, while our optimized construct design and efficient derivation strategy ensure the robust expression of the large transgenes. In combination with different methods for DNA methylation read-out, this system can now be used to study the role of the IDR and ROI in maintaining the steady-state level of DNA methylation against mechanisms of passive and active demethylation, but also for studying phenotypes affecting the efficiency of DNMT1 recruitment in the future

    Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea

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    ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy action’s effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (−10% per year) trend after adoption and enforcement of these laws (β2 = 0.18, p-value = 0.07; β3 = −0.10, p-value = 0.02). SHS exposure at home (β2 = 0.10, p-value = 0.09; β3 = −0.03, p-value = 0.14) and the primary cigarette smoking rate (β2 = 0.03, p-value = 0.10; β3 = 0.008, p-value = 0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK

    Comparative genomics of recent adaptation in Candida pathogens

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    [eng] Fungal infections pose a serious health threat, affecting >1,000 million people and causing ~1.5 million deaths each year. The problem is growing due to insufficient diagnostic and therapeutic options, increased number of susceptible patients, expansion of pathogens partly linked to climate change and the rise of antifungal drug resistance. Among other fungal pathogens, Candida species are a major cause of severe hospital-acquired infections, with high mortality in immunocompromised patients. Various Candida pathogens constitute a public health issue, which require further efforts to develop new drugs, optimize currently available treatments and improve diagnostics. Given the high dynamism of Candida genomes, a promising strategy to improve current therapies and diagnostics is to understand the evolutionary mechanisms of adaptation to antifungal drugs and to the human host. Previous work using in vitro evolution, population genomics, selection inferences and Genome Wide Association Studies (GWAS) have partially clarified such recent adaptation, but various open questions remain. In the three research articles that conform this PhD thesis we addressed some of these gaps from the perspective of comparative genomics. First, we addressed methodological issues regarding the analysis of Candida genomes. Studying recent adaptation in these pathogens requires adequate bioinformatic tools for variant calling, filtering and functional annotation. Among other reasons, current methods are suboptimal due to limited accuracy to identify structural variants from short read sequencing data. In addition, there is a need for easy-to-use, reproducible variant calling pipelines. To address these gaps we developed the “personalized Structural Variation detection” pipeline (perSVade), a framework to call, filter and annotate several variant types, including structural variants, directly from reads. PerSVade enables accurate identification of structural variants in any species of interest, such as Candida pathogens. In addition, our tool automatically predicts the structural variant calling accuracy on simulated genomes, which informs about the reliability of the calling process. Furthermore, perSVade can be used to analyze single nucleotide polymorphisms and copy number-variants, so that it facilitates multi-variant, reproducible genomic studies. This tool will likely boost variant analyses in Candida pathogens and beyond. Second, we addressed open questions about recent adaptation in Candida, using perSVade for variant identification. On the one hand, we investigated the evolutionary mechanisms of drug resistance in Candida glabrata. For this, we used a large-scale in vitro evolution experiment to study adaptation to two commonly-used antifungals: fluconazole and anidulafungin. Our results show rapid adaptation to one or both drugs, with moderate fitness costs and through few mutations in a narrow set of genes. In addition, we characterize a novel role of ERG3 mutations in cross-resistance towards fluconazole in anidulafungin-adapted strains. These findings illuminate the mutational paths leading to drug resistance and cross-resistance in Candida pathogens. On the other hand, we reanalyzed ~2,000 public genomes and phenotypes to understand the signs of recent selection and drug resistance in six major Candida species: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis and C. orthopsilosis. We found hundreds of genes under recent selection, suggesting that clinical adaptation is diverse and complex. These involve species-specific but also convergently affected processes, such as cell adhesion, which could underlie conserved adaptive mechanisms. In addition, using GWAS we predicted known drivers of antifungal resistance alongside potentially novel players. Furthermore, our analyses reveal an important role of generally-overlooked structural variants, and suggest an unexpected involvement of (para)sexual recombination in the spread of resistance. Taken together, our findings provide novel insights on how Candida pathogens adapt to human-related environments and suggest candidate genes that deserve future attention. In summary, the results of this thesis improve our knowledge about the mechanisms of recent adaptation in Candida pathogens, which may enable improved therapeutic and diagnostic applications.[cat] Les infeccions fúngiques representen una greu amenaça per a la salut, afectant a més de 1.000 milions de persones i causant aproximadament 1,5 milions de morts cada any. El problema està augmentant a causa d’unes opcions terapèutiques i diagnòstiques insuficients, l'increment del nombre de pacients susceptibles, l'expansió dels patògens parcialment vinculada al canvi climàtic i l'augment de la resistència als fàrmacs antifúngics. D’entre diversos fongs patògens, els llevats del gènere Candida són una causa important d'infeccions nosocomials, amb una alta mortalitat en pacients immunodeprimits. Diverses espècies de Candida constitueixen un problema de salut pública, cosa que requereix més esforços per a desenvolupar nous medicaments, optimitzar els tractaments disponibles i millorar els diagnòstics. Tenint en compte el dinamisme genòmic d’aquests patògens, una estratègia prometedora per millorar les teràpies i diagnòstics actuals és comprendre els mecanismes evolutius d'adaptació als fàrmacs antifúngics i a l’hoste humà. Treballs anteriors utilitzant l'evolució in vitro, la genòmica de poblacions, les inferències de selecció i els estudis d'associació de genoma complet (GWAS, per les sigles en anglès) han aclarit parcialment aquesta adaptació recent, però encara hi ha diverses preguntes obertes. En els tres articles que conformen aquesta tesi doctoral, hem abordat algunes d'aquestes preguntes des de la perspectiva de la genòmica comparativa. En primer lloc, hem abordat qüestions metodològiques relatives a l'anàlisi dels genomes de les espècies Candida. L'estudi de l'adaptació recent en aquests patògens requereix eines bioinformàtiques adequades per a la detecció, filtratge i anotació funcional de variants genètiques. Entre altres raons, els mètodes actuals són subòptims a causa de la limitada precisió per identificar variants estructurals a partir de dades de seqüenciació amb lectures curtes. A més, hi ha una necessitat d’eines computacionals per a la detecció de variants que siguin senzilles d'utilitzar i reproduibles. Per abordar aquestes mancances, hem desenvolupat el mètode bioinformàtic "personalized Structural Variation detection" (perSVade), una eina que permet la detecció, filtratge i anotació de diversos tipus de variants, incloent-hi les variants estructurals, directament des de les lectures. PerSVade permet la identificació precisa de les variants estructurals en qualsevol espècie d'interès, com ara els patògens Candida. A més, la nostra eina prediu automàticament la precisió de la detecció d’aquestes variants en genomes simulats, la qual cosa informa sobre la fiabilitat del procés. Finalment, perSVade es pot utilitzar per analitzar altres tipus de variants, com els polimorfismes de nucleòtid únic o els canvis en el nombre de còpies, facilitant així estudis genòmics integrals i reproduibles. Aquesta eina probablement impulsarà les anàlisis genòmiques en els patògens Candida i també en altres espècies. En segon lloc, hem abordat algunes de les preguntes obertes sobre l'adaptació recent en els llevats Candida, utilitzant perSVade per a la identificació de variants. D'una banda, hem investigat els mecanismes evolutius de resistència als fàrmacs antifúngics en Candida glabrata. Per a això, hem utilitzat un experiment d'evolució in vitro a gran escala per estudiar l'adaptació a dos antifúngics comuns: el fluconazol i l’anidulafungina. Els nostres resultats mostren una adaptació ràpida a un o ambdós fàrmacs, amb un cost per al creixement moderat i a través de poques mutacions en un nombre reduït de gens. A més, hem caracteritzat un paper nou de les mutacions en ERG3 en la resistència creuada al fluconazol en soques adaptades a anidulafungina. Aquests descobriments aclareixen els processos mutacionals que condueixen a la resistència als fàrmacs i a la resistència creuada en els patògens Candida. D'altra banda, hem re-analitzat aproximadament 2.000 genomes i fenotips disponibles en repositoris públics per a comprendre els senyals genòmics de selecció recent i de resistència a fàrmacs antifúngics, en sis espècies rellevants de Candida: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis i C. orthopsilosis. Hem trobat centenars de gens sota selecció recent, suggerint que l'adaptació clínica és diversa i complexa. Aquests gens estan relacionats amb funcions específiques de cada espècie, però també trobem processos alterats de manera similar en diferents patògens, com per exemple l’adhesió cel·lular, cosa que indica fenòmens d’adaptació conservats. A part, utilitzant GWAS hem predit mecanismes esperats de resistència a antifúngics i també possibles nous factors. A més, les nostres anàlisis revelen un paper important de les variants estructurals, generalment poc estudiades, i suggereixen una implicació inesperada de la recombinació (para)sexual en la propagació de la resistència. En conjunt, els nostres descobriments proporcionen noves perspectives sobre com els patògens Candida s'adapten als entorns humans, i suggereixen gens candidats que mereixen investigacions futures. En resum, els resultats d’aquesta tesi milloren el nostre coneixement sobre els mecanismes d'adaptació recent en els patògens Candida, cosa que pot permetre el disseny de noves teràpies i diagnòstics

    Studies on genetic and epigenetic regulation of gene expression dynamics

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    The information required to build an organism is contained in its genome and the first biochemical process that activates the genetic information stored in DNA is transcription. Cell type specific gene expression shapes cellular functional diversity and dysregulation of transcription is a central tenet of human disease. Therefore, understanding transcriptional regulation is central to understanding biology in health and disease. Transcription is a dynamic process, occurring in discrete bursts of activity that can be characterized by two kinetic parameters; burst frequency describing how often genes burst and burst size describing how many transcripts are generated in each burst. Genes are under strict regulatory control by distinct sequences in the genome as well as epigenetic modifications. To properly study how genetic and epigenetic factors affect transcription, it needs to be treated as the dynamic cellular process it is. In this thesis, I present the development of methods that allow identification of newly induced gene expression over short timescales, as well as inference of kinetic parameters describing how frequently genes burst and how many transcripts each burst give rise to. The work is presented through four papers: In paper I, I describe the development of a novel method for profiling newly transcribed RNA molecules. We use this method to show that therapeutic compounds affecting different epigenetic enzymes elicit distinct, compound specific responses mediated by different sets of transcription factors already after one hour of treatment that can only be detected when measuring newly transcribed RNA. The goal of paper II is to determine how genetic variation shapes transcriptional bursting. To this end, we infer transcriptome-wide burst kinetics parameters from genetically distinct donors and find variation that selectively affects burst sizes and frequencies. Paper III describes a method for inferring transcriptional kinetics transcriptome-wide using single-cell RNA-sequencing. We use this method to describe how the regulation of transcriptional bursting is encoded in the genome. Our findings show that gene specific burst sizes are dependent on core promoter architecture and that enhancers affect burst frequencies. Furthermore, cell type specific differential gene expression is regulated by cell type specific burst frequencies. Lastly, Paper IV shows how transcription shapes cell types. We collect data on cellular morphologies, electrophysiological characteristics, and measure gene expression in the same neurons collected from the mouse motor cortex. Our findings show that cells belonging to the same, distinct transcriptomic families have distinct and non-overlapping morpho-electric characteristics. Within families, there is continuous and correlated variation in all modalities, challenging the notion of cell types as discrete entities

    Evolutionäre Diversität der CLCA Gene zwischen Vögeln und Säugetieren

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    The chloride channel regulators, calcium activated (CLCA) gene family has mainly been associated with cancer and chronic inflammatory airway diseases but the presumably complex cellular functions of these gene products are still widely unknown. The family comprises four distinct genetic clusters in mammals, termed CLCA1 to CLCA4. It is highly complex and diverse and includes amplified or inactivated genes with a high degree of variation between species. For example, in contrast to mice with eight CLCAs including one inactivated gene, humans have only three intact CLCAs and one inactivated gene. The tissue and cellular expression patterns of different CLCA homologues within a species are also often redundant. This complexity and redundancy of the CLCA members might be a reason, why the function of this gene family has not been revealed yet based on a mammalian model organism. The complexity and redundancy of mammalian CLCAs raise two questions: Are the complexity and diversity of these genes unique features of mammals? And second, what is the evolutionary background of these peculiar developments? To address these questions, data on CLCA homologues obtained from evolutionarily more distant species are needed. Recently, a rather simple CLCA gene locus was predicted for the chicken, comprising only two CLCA genes. In a phylogenetic analysis, the first galline CLCA gene product, gCLCA1, was found closely related to mammalian CLCA1, 3 and 4. In contrast, the second one, gCLCA2, seemed more closely related to mammalian CLCA2 than to gCLCA1 or mammalian CLCA1, 3 and 4. In this cumulative thesis, the galline CLCA genes and their genomic structures were analyzed and both members were cloned. Their protein architecture and biochemical properties were investigated in silico and in vitro. In addition, their mRNA as well as their cellular protein expression patterns were analyzed. All data were compared to mammalian CLCA1, 2, 3, and 4. Both avian proteins are encoded by 14 exons and are located in a conserved locus between the Outer Dense Fiber of Sperm Tails 2-Like (ODF2L) and SH3-Domain GRB2-Like Endophilin B1 (SH3GLB1) genes. gCLCA1 combines many properties of mammalian CLCA1, 3 and 4 as it was shown to be a cleaved protein with a typical CLCA domain architecture. Despite its relatively high phylogenetic distance to mammalian CLCA4, it shares most common traits with this member. This includes heavy asparagine-linked glycosylation, the presence of a transmembrane domain in the carboxy-terminal cleavage product and protein expression in the apical membrane of enterocytes. In contrast, gCLCA2 was highly similar to mammalian CLCA2 in terms of its protein architecture, cleavage and glycosylation. These findings were in line with results from in silico analyses of CLCA2 sequences from other avian species.Furthermore, the presence of a transmembrane domain in the carboxy-terminal cleavage product and its expression in keratinocytes are traits of avian CLCA2, which are also found in mammalian CLCA2. Interestingly, and as opposed to the expression patterns of mammalian CLCA proteins, no overlapping tissue or cellular expression patterns were detected for the two galline CLCA members. Based on these findings, CLCA2 appears to be highly conserved among birds and mammals. The results allow to speculate that a hypothetical gene ancestor was expressed in keratinocytes of a common ancestral species before mammalian and avian lineages diverged. This high degree of CLCA2 conservation is in contrast to gCLCA1 and mammalian CLCA1, 3 and 4. During evolution, a hypothetical ancient ancestor of gCLCA1 / mammalian CLCA1, 3, and 4, was likely expressed by enterocytes of a common ancestral species of mammals and birds. The hypothetical ancestral gene seems to have expanded by gene duplications in the mammalian lineage, which did not occur in birds. Besides that, these findings cannot only be used to unveil the evolutionary history of the CLCA family but should be taken into account with regard to the selection of an animal model for the functional analysis of these genes. The chicken might serve as a promising species for knockout models to study CLCA2 functions in vivo. Results obtained from such a knockout chicken are likely transferable to mammalian CLCA2 due to the high degree of conservation. A chicken gCLCA1 knockout model might provide data, which might be transferred most likely to mammalian CLCA4 genes in the gut, as both share a similar cell type specific protein expression in this microenvironment, a similar protein architecture as well as similar biochemical properties. At the transition to the post-genomic era with publically accessible information on gene structures as well as nucleotide and protein sequences of various species, comprehensive analyses of gene families across species have become possible. The comparison of such data in combination with the comparison of gene related information, including cellular expression patterns and biochemical properties, is a powerful approach to enlighten the evolutionary background of proteins. Furthermore, it might be beneficial to identify the most suitable animal model for further functional and biomedical studies.Die CLCA, engl. „chloride channel regulator, calcium-activated“ Genfamilie wird hauptsächlich mit Krebserkrankungen sowie chronisch entzündlichen Atemwegserkrankungen in Zusammenhang gebracht. Die mutmaßlich komplexen Funktionen dieser Gene sind bisher jedoch noch weitgehend unbekannt. Die CLCA Genfamilie umfasst bei Säugetieren vier verschiedene Cluster, die als CLCA1 bis CLCA4 bezeichnet werden. Sie zeichnet sich durch eine außerordentliche Komplexität und Vielfältigkeit aus und beinhaltet tierartlich variierend amplifizierte und inaktivierte Gene. So besitzt der Mensch beispielsweise drei intakte CLCAs sowie ein inaktiviertes Gen, wohingegen die Maus über 8 CLCAs verfügt, von denen ebenfalls eines als inaktiviertes Gen vorliegt. Darüber hinaus sind die Gewebe- und Zellexpressionsmuster der CLCA-Homologen auch innerhalb einer Spezies häufig redundant. Diese Komplexität und Redundanz von CLCA könnten Gründe sein, welche die Aufdeckung der Funktion dieser Genfamilie im Säugetiermodell bisher erschwerte. Damit stellen sich zwei Fragen: Ist die Komplexität dieser Genfamilie eine Eigenart der Säugetiere? Und was ist der evolutionäre Hintergrund für diese Entwicklungen? Um diese Fragen zu beantworten, werden Daten über CLCA benötigt, die von evolutionär weiter entfernten Arten stammen. Kürzlich wurde für das Huhn ein relativ einfacher CLCA Genlokus vorhergesagt, welcher nur zwei CLCA Gene umfasst. Das erste CLCA Genprodukt des Huhns, gCLCA1, zeichnet sich durch eine besondere phylogenetische Nähe zu CLCA1, 3 und 4 der Säugetiere aus. Im Gegensatz dazu ist das zweite CLCA Genprodukt, gCLCA2, näher mit Säuger-CLCA2 verwandt als mit gCLCA1 oder Säuger-CLCA1, 3 oder 4. In dieser kumulativen These wurden die gallinen CLCA Gene sowie deren genomische Struktur analysiert und beide Homologe wurden kloniert. Darüber hinaus wurde deren Proteinarchitektur und die biochemischen Eigenschaften in silico und in vitro untersucht. Weiterhin wurden die mRNA- und zellulären Proteinexpressionsmuster im Vergleich zu CLCA1, 2, 3 und 4 bei Säugetieren analysiert. Beide Vogelproteine werden von 14 Exons kodiert und befinden sich an einem konservierten Ort zwischen den ODF2L, engl. „Outer Dense Fiber of Sperm Tails 2-Like” und SH3GLB1, engl „SH3-Domain GRB2-Like Endophilin B1“ Genen. gCLCA1 vereint mit seiner Proteinspaltung sowie der Proteinarchitektur viele Eigenschaften von CLCA1, 3 und 4 der Säugetiere. Trotz einer relativ großen phylogenetischen Entfernung zu Säuger-CLCA4 weist es jedoch die meisten gemeinsamen Merkmale mit diesem CLCA-Vertreter auf. Hierzu zählen eine starke, an Asparagin gekoppelte Glykosylierung, eine Transmembrandomäne im carboxyterminalen Spaltprodukt und eine Proteinexpression in der apikalen Membran von Enterozyten. Im Gegensatz dazu ist gCLCA2 dem Säugetier-CLCA2 in Bezug auf dessen Phylogenie, Proteinarchitektur, Spaltung und Glykosylierung sehr ähnlich. Diese Befunde ließen sich mittels in silico Analysen auch für weitere aviäre CLCAs nachweisen. Daneben sind die Präsenz einer Transmembrandomäne im carboxyterminalen Spaltprodukt und die Expression in Keratinozyten Merkmale des aviären CLCA2, die auch bei Säugetier-CLCA2 vorzufinden sind. Bemerkenswerterweise fanden sich im Gegensatz zu den Säuger-CLCAs bei den gallinen CLCA-Mitgliedern keine überlappenden gewebe- und zellspezifischen Expressionsmuster. Unter Betrachtung dieser Befunde erscheint CLCA2 bei Vögeln und Säugetieren stark konserviert zu sein. Weiterhin lässt sich auf Basis der in dieser kumulativen These erhobenen Daten spekulieren, dass ein hypothetischer gemeinsamer genetischer Vorfahre wahrscheinlich in Keratinozyten eines gemeinsamen Vorfahren von Vögeln und Säugern exprimiert wurde. Dieser hohe Grad an Konservierung von CLCA2 steht im Gegensatz zu demjenigen von gCLCA1 und Säugetier-CLCA1, 3 und 4. Im Laufe der Evolution scheint ein hypothetischer genetischer Vorfahre von gCLCA1/Säugetier-CLCA1, 3 und 4 vermutlich in Enterozyten eines gemeinsamen Vorfahren von Vögeln und Säugern exprimiert worden zu sein. Dieser hypothetische genetische Vorfahre scheint durch Genduplikationen bei Säugetieren expandiert zu sein, nicht jedoch bei Vögeln. Neben diesen Aussagen zu einem wahrscheinlichen evolutionären Szenario der CLCA Genfamilie zwischen Vogel und Säuger lassen sich die Ergebnisse jedoch auch zur Auswahl eines Modellorganismus zur funktionalen Analyse dieser Gene nutzen. Hierbei erscheint das Huhn zur Untersuchung der CLCA2-Funktion in vivo ein vielversprechender Kandidat für knockout-Modelle, deren Untersuchungsergebnisse durch den Grad an Konservierung mit hoher Wahrscheinlichkeit auf Säugetiere übertragbar sind. Ein gCLCA1-Knockoutmodell könnte hingegen Daten liefern, die hochwahrscheinlich auf CLCA4 Gene von Säugetieren im Darm übertragbar sind, da beide in dieser anatomischen Lokalisation ein vergleichbares zellspezifisches Expressionsmuster sowie eine ähnliche Proteinarchitektur und biochemische Eigenschaften besitzen. Im Anbruch des postgenomischen Zeitalters, in dem Genstrukturen sowie Nukleotid- und Proteinsequenzen verschiedener Spezies öffentlich leicht zugänglich sind, werden umfassende, vergleichende Analysen von Genfamilien über verschiedene Spezies hinweg möglich. In Kombination mit dem Vergleich von genbezogenen Daten wie zellulären Expressionsmustern oder biochemischen Eigenschaften der Genprodukte ist dies ein wirkungsvolles Vorgehen, um den evolutionären Hintergrund von Proteinen aufzudecken und das geeignetste Tiermodell für weitere wissenschaftliche Fragestellung auszuwählen

    Molecular signals of arms race evolution between RNA viruses and their hosts

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    Viruses are intracellular parasites that hijack their hosts’ cellular machinery to replicate themselves. This creates an evolutionary “arms race” between hosts and viruses, where the former develop mechanisms to restrict viral infection and the latter evolve ways to circumvent these molecular barriers. In this thesis, I explore examples of this virus-host molecular interplay, focusing on events in the evolutionary histories of both viruses and hosts. The thesis begins by examining how recombination, the exchange of genetic material between related viruses, expands the genomic diversity of the Sarbecovirus subgenus, which includes SARS-CoV responsible for the 2002 SARS epidemic and SARS-CoV-2 responsible for the COVID-19 pandemic. On the host side, I examine the evolutionary interaction between RNA viruses and two interferon-stimulated genes expressed in hosts. First, I show how the 2′-5′-oligoadenylate synthetase 1 (OAS1) gene of horseshoe bats (Rhinolophoidea), the reservoir host of sarbecoviruses, lost its anti-coronaviral activity at the base of this bat superfamily. By reconstructing the Rhinolophoidea common ancestor OAS1 protein, I first validate the loss of antiviral function and highlight the implications of this event in the virus-host association between sarbecoviruses and horseshoe bat hosts. Second, I focus on the evolution of the human butyrophilin subfamily 3 member A3 (BTN3A3) gene which restricts infection by avian influenza A viruses (IAV). The evolutionary analysis reveals that BTN3A3’s anti-IAV function was gained within the primates and that specific amino acid substitutions need to be acquired in IAVs’ NP protein to evade the human BTN3A3 activity. Gain of BTN3A3-evasion-conferring substitutions correlate with all major human IAV pandemics and epidemics, making these NP residues key markers for IAV transmissibility potential to humans. In the final part of the thesis, I present a novel approach for evaluating dinucleotide compositional biases in virus genomes. An application of my metric on the Flaviviridae virus family uncovers how ancestral host shifts of these viruses correlate with adaptive shifts in their genomes’ dinucleotide representation. Collectively, the contents of this thesis extend our understanding of how viruses interact with their hosts along their intertangled evolution and provide insights into virus host switching and pandemic preparedness

    Unlocking the genome of perch - From genes to ecology and back again

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    Eurasian perch Perca fluviatilis has been a popular model species for decades in the fields of aquatic ecology, community dynamics, behaviour, physiology and ecotoxicology. Yet, despite extensive research, the progress of integrating genomic perspective into existing ecological knowledge in perch has been relatively modest. Meanwhile, the emergence of high-throughput sequencing technologies has completely changed the methods for genetic variation assessment and conducting biodiversity and evolutionary research. During the last 5 years, three genome assemblies of P. fluviatilis have been generated, allowing substantial advancement of our understanding of the interactions between ecological and evolutionary processes at the whole-genome level. We review the past progress, current status and potential future impact of the genomic resources and tools for ecological research in Eurasian perch focusing on the utility of recent whole-genome assemblies. Furthermore, we demonstrate the power of genome-wide approaches and newly developed tools and outline recent cases where genomics have contributed to new ecological and evolutionary knowledge. We explore how the availability of reference assembly enables the efficient application of various statistical tools, and how genomic approaches can provide novel insights into resource polymorphism, host-parasite interactions and to genetic and phenotypic changes associated with climate change and harvesting-induced evolution. In summary, we call for increased integration of genomic tools into ecological research for perch, as well as for other fish species, which is likely to yield novel insights into processes linking the adaptation and plasticity to ecosystem functioning and environmental change

    Interplay of genetic, epigenetic and transcription factors in the regulation of transcriptional variation in Plasmodium falciparum

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    [eng] The most severe form of malaria, caused by Plasmodium falciparum parasites, still kills over half a million people every year, most of them children under the age of five. Despite huge research efforts, reduction in the global burden of disease has stalled in recent years. P. falciparum has a very complex life cycle including, among other steps, sexual reproduction in female Anopheles mosquitos and an asexual intra-erythoricitic development cycle (IDC) inside the human host, which causes the disease. During the IDC, the parasite needs to continuously adapt to changes in its environment including fluctuations in blood temperature, concentration of nutrients and other metabolites, presence of drugs, and a constant fight against the host’s immune system. In this thesis, we have studied the adaptation mechanisms of P. falciparum to this plethora of challenges, with a special focus on clonally variant genes (CVGs). In P. falciparum, CVGs are a set of genes, participating in host-parasite interactions, which can be found both in a transcriptionally active state, characterized by euchromatin, or a transcriptionally silenced state, characterized by heterochromatin. The state of CVGs is inherited by the progeny of a parasite, with stochastic switches occurring at a low frequency. Parasites with the most optimal patterns of CVGs expression are continuously selected as the environment changes, leading to adaptation and survival of the infecting population. In the first paper of this thesis, we have analyzed subcloned parasite populations to characterize, with unprecedented detail, the heterochromatin distribution associated with the active and silenced states of CVGs. This has allowed us to define different kinds of heterochromatin transitions between the active and silenced states of CVGs and has given us new insights on the regulation of var genes (one of the main virulence factors for malaria) and into the regulation of sexual conversion, a process crucial for malaria transmission. Continuing with CVG regulation, in the second paper of the thesis, we have analyzed how patterns of CVG expression are established at the onset of human infections, after passage through transmission stages. Our results suggest a loss of the epigenetic memory during transmission stages and a reset of the heterochromatin patterns that drive CVG expression. Similar patterns of CVG expression arose in different infected individuals, suggesting that the activation probability of a given CVG is an intrinsic property of the gene. In the third paper of the thesis, we have further studied the sexual conversion phenomenon. We have generated a conditional over-expression system for pfap2-g, the CVG that acts as master regulator of sexual conversion, achieving sexual conversion rates of ~90% after induction. Our results have provided new insights on how heterochromatin at different positions affects expression of pfap2-g and have allowed us to characterize the transcriptional profile of the initial stages of sexual commitment with unprecedented sensitivity. Finally, in the fourth paper of this thesis, we have studied the adaptation of the parasite to heat-shock, which happens in natural infections due to fever episodes. We expected CVGs to participate in this phenomenon, but instead we have identified pfap2-hs, a non-clonally variant transcription factor (TF), as the main driver of the heat-shock response in P. falciparum. AP2-HS acts as the functional homolog of HSF1 (a TF that drives the heat-shock response from yeast to mammals, but is absent in P. falciparum), driving a very tight transcriptional response to heat-shock, characterized by the up-regulation of hsp70 and hsp90. Although the presence of directed responses had previously been demonstrated for other cues, it is the first time that the transcription factor driving such a response is identified in P. falciparum. Taken together, the results of this thesis have broadened our knowledge of the regulation of adaptive mechanisms in P. falciparum. Learning about this deadly parasite’s defense mechanisms will be instrumental to design better strategies to fight it back in the future
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