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A Temporal Analysis of Host Chromatin Changes and Transcriptional Responses Induced by Latent Kaposi’s Sarcoma associated Herpesvirus Infection
No full textThe human ɣ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with several cancers, including Kaposi Sarcoma, a tumor of endothelial origin as well as the two B cell malignancies Primary Effusion Lymphoma and Multicentric Castleman’s Disease. Like other herpesviruses, KSHV can establish a latent state of infection, which allows life-long persistence in its host. Previous studies revealed that latent KSHV infection does not only lead to the acquisition of epigenetic modifications on the viral genome but might also induce re-localization of epigenetic host factors. Additionally, there is evidence from more recent data that KSHV infected cells undergo epigenetic changes at distinct host loci which might play a role during tumorigenesis. However, a detailed and, more importantly, a temporal genome wide analysis of host epigenetic changes upon KSHV infection in a relevant in vitro system is still missing.
Thus, the aim of this study was to investigate KSHV-induced epigenetic short- and long-term consequences with the help of an efficient latent infection system in endothelial cells. ChIP-seq, MeDIP-seq and RNA-seq were performed to monitor histone modification and DNA methylation changes, as well as associated alterations of transcription patterns at various time points post infection.
Despite the moderate transcriptomic deregulation observed during KSHV infection, a distinct interferon response signature persisted for several weeks in strictly latent cultures. Single cell RNA-seq furthermore suggests that these signatures originated from a small subset of cells, which show no evidence of lytic reactivation. While interferon regulated genes were not affected by histone modification changes due to a potential epigenetic pre-activation, several other regions were identified that undergo significant epigenetic remodeling. Remodeling was observed for activating as well as repressive histone modifications, with the most evident changes occurring at the level of acetylated histone H3 lysine 27 (H3K27ac), which is a mark for active genes as well as enhancer regions. Multiple altered regions mapped to genes which exhibited mild but detectable transcriptional deregulation. However, remodeled regions were also detected which are not directly linked to differentially expressed genes, indicating a potential predisposition which might only lead to phenotypic manifestation upon the presence of additional stimuli. Comparison of KSHV infected and cleared cells allowed for the investigation of such predisposed regions. A subset of histone modification changes remained stable in the cleared population although the transcriptional profile of these cells was almost completely reconstituted to the initial uninfected state. Challenging these cells with different stimuli, like KSHV re-infection led to a more pronounced transcriptional response of at least some of the predisposed regions.
Based on these findings, we suspect that KSHV actively modulates epigenetic pathways, which might affect the viral life cycle, but also lead to alterations of the host epigenome. Beside KSHV dependent chromatin changes, a subset of deregulated regions might become independent of the infection as indicated by stable changes in KSHV cleared cells. As it is thought that virus-mediated tumorigenesis is a long-term consequence of latent infection, these distinct epigenetic states, potentially together with other stimuli, may promote KSHV mediated tumorigenesis.Das ɣ-Herpesvirus Kaposi Sarkom-assoziiertes Herpesvirus (KSHV) ist ein humanpathogenes Virus, welches mit der Entstehung zahlreicher Tumore assoziiert ist. Dazu zählt zum einen das Kaposi Sarkom, ein Tumor endothelialen Ursprungs, sowie die beiden B Zell Tumore Primäres Effusionslymphom und die multizentrische Castleman Erkrankung. Wie auch andere Herpesviren kann KSHV einen latenten Infektionsstatus einnehmen, der es ihm erlaubt lebenslang im Wirt zu persistieren. Während dieses latenten Lebenszyklus erhält das virale Genom nicht nur epigenetische Modifikationen, um große Teile seines Genoms transkriptionell still zu legen. KSHV scheint auch in der Lage zu sein, epigenetische Faktoren gezielt zu manipulieren, um den Aufbau der epigenetischen Modifikationen auf dem viralen Genom zu fördern. Neuere Erkenntnisse deuten ebenfalls darauf hin, dass KSHV infizierte Zellen epigenetische Veränderungen in spezifischen Wirtsregionen aufweisen, die eine Rolle bei der Virus-induzierten Tumorentstehung spielen könnten. Allerdings wurde bezüglich dieser veränderten Regionen bisher keine detaillierte und zeitlich aufgelöste Genom-weite Studie in einem relevanten in vitro System durchgeführt.
Das Ziel dieser Arbeit war es deshalb, ein latentes KSHV Infektionssystem in Endothelzellen zu etablieren, womit die Analyse von KSHV induzierten Kurz-und Langzeitfolgen auf dem Wirtsepigenom ermöglicht wurde. ChIP-seq, MeDIP-seq und RNA-seq wurden durchgeführt, um Veränderungen von Histonmodifikationen und DNA-Methylierung zu detektieren und diese auch mit transkriptionellen Mustern zu korrelieren.
Obwohl lediglich geringe transkriptionelle Veränderungen während einer KSHV Infektion festgestellt wurden, war dennoch eine deutliche Interferon Antwort in latent infizierten Zellen erkennbar, die sich über einen unerwartet langen Zeitraum erstreckte. Mithilfe von Einzel-Zell Sequenzierung konnte darüber hinaus gezeigt werden, dass lediglich einzelne Zellen für diese späte Immunantwort verantwortlich sind, die allerdings keinerlei Zeichen einer lytischen Reaktivierung von KSHV aufwiesen. Während für Interferon-induzierte Gene kaum Veränderungen auf Histonmodifikationsebene festgestellt werden konnten, womöglich aufgrund eines epigenetisch prä-aktivierten Zustands, wurden zahlreiche andere Regionen mit differentiellen epigenetischen Mustern identifiziert. Veränderungen konnten sowohl für aktivierende also auch für reprimierende Modifikationen detektiert werden, jedoch zeigten sich die am auffälligsten veränderten Regionen fast immer auf der Ebene von H3K27ac, eine Modifikation, die charakteristisch für Enhancer-Bereiche ist. Während zahlreiche dieser differentiellen Bereiche auch leichte, aber detektierbare Veränderungen bezüglich der Transkription aufwiesen, gab es auch einige Regionen, die nicht direkt mit einer transkriptionellen Antwort einhergingen. Deshalb wurde vermutet, dass solche Regionen einer Prädisposition unterzogen werden, die lediglich durch einen sekundären Stimulus zu einer phänotypischen Ausprägung führt. Um diese Hypothese zu untersuchen wurden KSHV infizierte Zellen mit einer parallel kultivierten Population verglichen, die eine Infektion erfolgreich überwinden konnte. Dies ermöglichte die Identifizierung von stabilen Veränderungen, die selbst nach einer abgeklungenen Infektion noch erhalten blieben und das obwohl auf transkriptioneller Ebene eine komplette Rekonstitution des initialen, nicht infizierten Zustands auftrat. Wurden diese Zellen mit unterschiedlichen Stimuli induziert, wie zum Beispiel einer Re-Infektion mit KSHV, konnte zumindest für einen Teil der Gene eine deutlich stärkere transkriptionelle Antwort im Vergleich zu den Kontrollzellen festgestellt werden.
Diese Daten deuten darauf hin, dass KSHV aktiv epigenetische Faktoren manipuliert, was zum einen den viralen Lebenszyklus beeinflusst, jedoch auch zu epigenetischen Veränderungen der Wirtszelle führt. Die Untersuchung der Zellen mit abgeklungener KSHV Infektion lassen die Vermutung zu, dass es neben KSHV abhängigen Veränderungen auch einen Anteil an deregulierten Regionen gibt, die Infektions-unabhängig werden. Beide Veränderungen könnten bei der KSHV abhängigen Tumorentstehung eine wichtige Rolle einnehmen, da dieser Prozess als Konsequenz einer latenten Langzeit-Infektion verstanden wird
The Alpha and the Gamma of latency establishment: Deciphering early events after nuclear entry of herpesvirus genomes
No full textHerpesviruses are pathogens that can infect humans and animals and are well adapted to their host. Primary infection with herpesviruses results in lifelong persistence of the herpesvirus, with the possibility of reactivation. After a first phase of lytic replication, most herpesviruses enter a state known as latency. During latency, very few viral genes are transcribed and the genomes can remain in the infected cell without being eliminated by the immune system. During latency, the herpesviral deoxyribonucleic acid (DNA) is modified by epigenetic modifications, i.e. DNA methylation and posttranslational repressive histone modifications. A recent study showed that the cytidine guanosine dinucleotide (CpG)-rich genome of the gammaherpesvirus Kaposi’s Sarcoma-associated herpesvirus (KHSV) rapidly attracts Polycomb repressive complexes (PRCs) via the non-canonical (nc) recruitment pathway. This mechanism normally serves to silence CpG islands of cellular DNA via recognition of unmethylated CpG motifs. Interestingly, alphaherpesviruses such as herpes simplex virus-1 (HSV-1) also have a very high content of CpG sequences. Previous results from our group suggest that PRC-mediated repression may be a default pathway to repress CpG-rich viral DNA (vDNA).
Therefore, we hypothesize that herpesviruses with high CpG content might generally exploit this pathway to support latency. Therefore, the study presented here aims to further validate this hypothesis. To this end, a direct comparative analysis was performed between KSHV and lytic-cycle-deficient mutants of the alphaherpesviruses HSV-1 and pseudorabies virus, which also have a very high CpG content. Genome-wide chromatin analyses were performed by chromatin-immunoprecipitation-sequencing (ChIP-seq), early after infection, to elucidate the epigenome of the lytic-cycle-deficient alphaherpesviruses. Furthermore, a fluorescence-based visualization method, which makes use of bioorthogonal chemistry, was utilized to track the spatiotemporal interaction of individual viral episomes with host factors that might be involved in herpesvirus silencing. In addition, knock-down of the host factor ATRX was performed with subsequent infection experiments to investigate its impact on herpesvirus reactivation.
The data presented here show for the first time rapid acquisition of the PRC-associated facultative heterochromatin mark H2AK119ub by lytic-cycle-deficient pseudorabies virus (PRV) and HSV-1 genomes. Interestingly, both viruses also acquired constitutive heterochromatin, via H3K9me3. Further analysis of the underlying mechanism revealed that the acquisition of the different forms of heterochromatin appears to be dependent on the infected cell type and thus very likely on distinct host factors. In particular, evidence was provided that the host factor Alpha-thalassemia/mental retardation syndrome X-linked (ATRX), a component of promyelocytic leukemia-nuclear bodies (PML-NBs), plays a role in the establishment of a tightly repressed condition of the virus genome and might influence constitutive heterochromatin formation.
These results suggest that alphaherpesvirus genomes, similar to KSHV, can attract PRCs via the ncPRC-recruitment pathway. Unlike latently replicating KSHV episomes, non-replicating alphaherpesvirus genomes did not acquire H3K27me3. Since H3K27me3 is preferentially formed on replicated DNA, the lack of vDNA replication in latent alphaherpesviruses may be related to the fact that H3K27me3 could not be detected on these genomes. Hence, vDNA replication may play an important role during the acquisition of the different forms of heterochromatin of herpesvirus genomes. Moreover, the data presented in this dissertation suggest a model in which alphaherpesviruses might establish two differentially silenced subpopulations. In this model, PRC-mediated repression leads to the establishment of authentic alphaherpesvirus latent reservoirs with the ability to reactivate, whereas PML-NB-mediated repression can lead to permanent silencing and, in dividing cells, even to subsequent elimination of the virus.
Together, these results show a new aspect of alphaherpesvirus heterochromatinization and partly generalize the PRC-mediated repression mechanism among two herpesvirus subfamilies. Therefore, this study enhances the understanding of the early repression of incoming herpesviral DNA
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Untersuchung des Einflusses des Kaposi Sarkom-assoziierten Herpesvirus auf Histonmodifikationsmuster der Wirtszelle
No full textDas Kaposi-Sarkom-Herpesvirus ist das ätiologische Agens diverser humaner Tumorerkrankungen. Es besitzt einen biphasischen Lebenszyklus. Während der lytischen Phase wird das virale Genom vollständig exprimiert und neue Viruspartikel gebildet. In der latenten Phase hingegen wird nur eine kleine Teilmenge viraler Gene exprimiert, sodass das Virus unerkannt von der Immunantwort des Wirts persistieren kann. Epigenetische Mechanismen, insbesondere Histonmodifikationen, scheinen eine wesentliche Rolle bei der Regulation der Transkription während der Latenz zu
spielen. Mangels eigener Proteine zur Etablierung von Histonmodifikationen muss
das Virus auf zelluläre Proteine zurückgreifen. Dies führt zu der Hypothese, dass
diese Interaktionen die zellulären Histonmodifikationen und damit potentiell die
Transkription beeinflussen können.
Die Untersuchung der Änderungen von Transkription und Histonmodifikationen in
Folge einer de novo KSHV-Infektion wurde in HDF- sowie in MC116-Zellen
durchgeführt. In HDFs zeigte sich, dass Differenzen in der Transkription im
Wesentlichen auf zelluläre Reaktionen auf die Infektion zurückgehen und über die
Zeit abnehmen. Auch für die Histonmodifikationen ließen sich nur wenige
Änderungen feststellen. Interessantester Ansatzpunkt war die Anreicherung der
H3K27me3 am TSHZ2-Gen, einem potentiellen Tumorsuppressor. Des Weiteren ließ
sich trotz der Korrelation von Histonmodifikationen und Transkription keine
Schnittmenge bei den jeweils detektierten Änderungen feststellen.
Wie in den HDF-Zellen zeigten sich auch für die MC116 nur wenige Änderungen in
Transkription und Histonmodifikationen. Jedoch konnte hier für zwei Gene sowohl für
die Transkription als auch die Histonmodifikationen signifikante Änderungen
festgestellt werden. Die genauere Untersuchung eines dieser Gene (TNFRSF1b)
ergab jedoch, dass daraus keine funktionalen Konsequenzen resultierten. Wie sich
zeigte hatten die Veränderungen keinen Einfluss auf den Protein-Level. Dies legt den
Schluss nahe, dass die Zelle die Änderungen der Transkription über regulatorische
Mechanismen kompensiert. Weitere interessante Differenzen der
Histonmodifikationen, jedoch ohne Änderung der Transkription, zeigten die Gene
EGFR, FGF12 und IGF1R. Die Änderungen könnten Prädispositionen für spätere
Expressionsänderungen darstellen und stellen somit interessante Ansatzpunkte für
weitere Untersuchungen dar.Kaposi’s sarcoma-associated herpesvirus is the etiologic agent of several human
tumors. It has a biphasic lifecycle, which is divided in the latent- and the lytic phase.
During the latter the entire viral genome is expressed and new viral particles are
produced. In contrast, during latency only a small subset of viral genes is expressed,
such that the virus can persist without inducing the host’s immune response.
Epigenetic Modifications, especially histone-modifications, play an important role in
the regulation of latent gene expression. Since the viral genome does not code for
proteins which directly establish or erase these modifications, the virus must interact
with cellular proteins to induce chromatin changes. These interactions could lead to
changes in cellular histone-modification levels and potentially to changes in gene
transcription.
For the investigation of the impact of de novo KSHV infection on transcription and
histone-modifications, human dermal fibroblasts (HDF) and MC116 (B-cell-
lymphoma) were used. In HDF-cells, changes in transcription seem to be primarily
caused by the cellular reaction to the infection and diminish over time. Also, there
were only few changes in the levels of histone-modifications. The most interesting of
these differential genes is TSHZ2, which is a possible tumor-suppressor and showed
enriched levels of H3K27me3. Furthermore, no correlation between changes in
transcription and changes in histone-modifications could be detected, although in
general transcription and histone modifications showed the expected correlation.
Like in HDF-cells, only few changes in transcription and histone modifications could
be detected for the MC116-cells. However, significant changes of both transcription
and histone-modifications could be detected for two genes. Investigation of one of
these genes (TNFRSF1b) showed no functional consequences of the detected
changes. Analysis of the protein levels showed no changes in TNFRSF1b-levels.
Therefore the cells somehow seem to compensate the changes in transcription via
other regulatory mechanisms. Further interesting genes which showed differential
histone-modification-levels were EGFR, FGF12 and IGF1R. Although these did not
show any changes in transcription, the changes in histone-modification-levels could
act as a predisposition for later changes in transcription. Therefore these genes are
of high interest for future investigations
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Osmotic Stress Interferes with DNA Damage Response and H2AX Phosphorylation in Human Keratinocytes
No full textThe human skin and in particular its outermost layer, the epidermis, protects the body from potentially harmful substances, radiation as well as excessive water loss. However, the interference between the various stress responses of the epidermal keratinocytes, which often occur simultaneously, is largely unknown. The focus of this study was to investigate the interference between osmotic stress and DNA damage response. In addition to revealing the already well-described regulation of diverse gene sets, for example, cellular processes such as transcription, translation, and metabolic pathways (e.g., the KEGG citrate cycle and Reactome G2/M checkpoints), gene expression analysis of osmotically stressed keratinocytes revealed an influence on the transcription of genes also related to UV-induced DNA damage response. A gene network regulating the H2AX phosphorylation was identified to be regulated by osmotic stress. To analyze and test the interference between osmotic stress and DNA damage response, which can be triggered by UV stress on the one hand and oxidative stress on the other, in more detail, primary human keratinocytes were cultured under osmotic stress conditions and subsequently exposed to UV light and H2O2, respectively. γH2AX measurements revealed lower γH2AX levels in cells previously cultured under osmotic stress conditions
Development of a software for the identification of novel and known pathogens in clinical samples
No full textDie Sequenzierung von diagnostischen Proben gilt als eine Schlüsseltechnologie, welche die Diagnostik von Infektionskrankheiten grundlegend verbessern kann. Mit diesem Ansatz können nicht nur Infektionserreger identifiziert werden, von denen bereits bekannt ist, dass sie mit einer bestimmten Krankheit assoziiert sind, sondern auch solche, welche bisher nicht mit dieser Krankheit in Verbindung gebracht wurden. Zudem ist der Ansatz geeignet vollständig unbekannte Infektionserreger zu identifizieren. Er kann daher dazu beitragen, zukünftigen Ausbrüchen neuartiger Infektionserreger besser vorbereitet zu begegnen.
Diese Studie beschreibt die Entwicklung und Anwendung von Methoden für die Identifizierung von Infektionserregern in diagnostischen Proben. Die Methoden wurden mehrfach erfolgreich unter klinischen Bedingungen eingesetzt und die entsprechenden Ergebnisse wurden im Rahmen dieser Doktorarbeit publiziert.
Die entwickelten Methoden stehen der wissenschaftlichen Gemeinschaft in Form einer quelloffenen Software zur Verfügung.
Unter Verwendung klinischer Proben wurde die neue Software mit Methoden konventioneller Diagnostik validiert und mit etablierten bioinformatischen Analyse-Pipelines verglichen. Sie detektiert Infektionserreger aus verschiedenen diagnostischen Entitäten zuverlässig und klassifiziert virale Erreger oft bis zur Ebene des Stammes. Darüber hinaus ist die Software in der Lage virale Genome vollständig zu rekonstruieren. Dies gelingt sogar in Proben, welche mit mehreren nah verwandten Stämmen infiziert sind.
Zusätzlich zu einer verbesserten Methode der taxonomische Klassifikation, bietet die neue Software auch Funktionen, welche in etablierten Analyse-Pipelines nicht vorhanden sind. Sie ist beispielsweise in der Lage, Proteindomänen zu annotieren und sie kann Sequenzen anhand dieser Annotationen klassifizieren. Als konservierte, funktionale Einheiten, können Proteindomänen zusätzliche Evidenz für das Vorhandensein möglicher Infektionserreger bieten. Sie können insbesondere dabei helfen, neuartige Infektionserreger zu identifizieren.
Neben der Auswertung einzelner Proben kann die Software auch kohortenbasierte Analysen durchführen. In diesem Modus werden probenübergreifende Vergleiche durchgeführt, um Sequenzsignaturen zu identifizieren welche in einer Gruppe von Proben im Vergleich zu einer Kontrollgruppe überrepräsentiert sind. Dieser Ansatz erfordert weder eine vorangegangene taxonomische Zuordnung, noch Homologie zu bereits beschriebenen Sequenzen. Er ermöglicht somit den Nachweis gänzlich neuartiger Infektionserreger.Sequencing of diagnostic samples is widely considered a key technology that may fundamentally improve infectious disease diagnostics. The approach can not only identify pathogens already known to cause a specific disease, but may also detect pathogens that have not been previously attributed to this disease, as well as completely new, previously unknown pathogens. Therefore, it may significantly increase the level of preparedness for future outbreaks of emerging pathogens.
This study describes the development and application of methods for the identification of pathogenic agents in diagnostic samples. The methods have been successfully applied multiple times under clinical conditions. The corresponding results have been published within the scope of this thesis. Finally, the methods were made available to the scientific community as an open source bioinformatics tool.
The novel software was validated by conventional diagnostic methods and it was compared to established analysis pipelines using authentic clinical samples. It is able to identify pathogens from different diagnostic entities and often classifies viral agents down to strain level. Furthermore, the method is capable of assembling complete viral genomes, even from samples containing multiple closely related viral strains of the same viral family.
In addition to an improved method for taxonomic classification, the software offers functionality which is not present in established analysis pipelines. It is, for example, able to annotate protein domains and it performs the classification of sequences based on these annotations. The conserved, functional domains provide an additional level of evidence for the presence of putatively pathogenic agents and they may aid especially the detection of novel pathogens.
Asides from the analysis of individual samples, the software can perform cohort-based analyses. In this mode cross-sample comparisons are carried out to identify sequence signatures which are overrepresented in a group of samples in comparison to a control group. This approach neither requires previous taxonomic classification nor sequence homology searches in external databases and thus enables the detection of truly novel pathogenic agents
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