Establishing a screen for enhancers active in Drosophila embryogenesis

Enhancer sind massgeblich an der Regulation zeitlicher und raumlicher Genexpression beteiligt. Ein starker Indikator für die Relevanz von Enhancern ist die Beobachtung, dass die DNA-Sequenz eines Enhancers gekoppelt an ein Reportergen zu einem gewebe-spezifischen Expressionsmuster dieses Gens führt. Die essentiellen Teile einer Enhancer Sequenz die dessen Funktion zugrunde liegen sind jedoch noch nicht bekannt. Um dieses Problem genauer zu ergründen, ist die Analyse einer grossen Anzahl an systematisch verifizierten Enhancern nötig. Da sich die bisherige Forschung jedoch weitgehend mit regulatorischen Regionen einzelner Gene in unterschiedlichen experimentellen Versuchsanordnungen und Organismen beschaftigt hat, existiert eine derartige Ressource noch nicht. Daher stellen wir eine Methode zur systematischen Identifizierung von embryonalen Enhancern in der Taufliege Drosophila Melanogaster vor. Insgesamt haben wir 981 potentielle Enhancer mithilfe von Reporter-Assays auf ihre zeit- und gewebsspezifische Aktivität hin untersucht. Eine unserer zentralen Beobachtungen zeigte einen graduellen Anstieg der Zahl aktiver Enhancer mit foranschreitender Embryonalentwicklung. Diese Beobachtung lasst sich durch Arbeiten anderer Forschungsgruppen erklaren, wonach im Laufe der Embryogenese sowohl die Zahl der Gewebe als auch die Komplexitat des Genexpressionsmuster in den einzelnen Geweben zunimmt. Der dadurch enstehende Bedarf an zusatzlicher Regulation erklart den beobachteten Anstieg aktiver Enhancer in spaten Embryogenese-Stadien. Um eine weitergehende Analyse unserer untersuchten aktiven Enhancer, welche unterschiedliche zeit- und gewebsspezifische Aktivitatsmuster zeigen, zu ermöglichen, wurde von uns ein computergestütztes Bildanalyseverfahren entwickelt. Dadurch konnten unsere Proben automatisiert auf zeitliche und raumliche Aktivitat analysiert und visuell dargestellt werden. Zusammenfassend stellen unsere Resultate eine zuverlassige Ausgangsbasis für weitere Analysen dar die in der Zukunft zum genaueren Verstandnis des zentralen Zusammenhangs zwischen der Sequenz und der Aktivitat eines Enhancers beitragen können.Enhancers are critical determinants of spatio-temporal gene expression. An enhancer's sole DNA sequence, cloned upstream of a reporter gene, is sufficient to drive expression which partially or fully resembles the endogenous gene's pattern. The sequence determinants which give rise to this specific activity are, however, unclear. While previous resarch concentrated on dissecting regulatory regions of individual genes, a large-scale approach might be needed to find shared sequences among enhancers with similar activity and thereby improve our understanding of the regulatory code. This thesis reports on a method for the identification and analysis of enhancers active in the embryogenesis of the common fruit fly Drosophila Melanogaster. Upon screening 981 enhancer candidates using an in-vivo reporter assay, we find 403 (41\%) to be active in at least one stage of development. Additionally, we find the fraction of active elements to increase as the developing embryo becomes more complex over time. For further analysis, we developed a computational pipeline enabling us to find elements of similar spatio-temporal activity and visualize their pattern over time and space. Overall, our results provide a reliable basis for future analysis which may lead to the identification of sequence elements determining an enhancer's specific activity

cis-Regulatory Requirements for Tissue-Specific Programs of the Circadian Clock

SummaryBackgroundBroadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs CLOCK (CLK) and CYCLE (CYC) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how CLK and CYC determine tissue-specific clock programs has remained unclear.ResultsHere, we use a functional genomics approach to determine the cis-regulatory requirements for clock specificity. We first determine CLK and CYC genome-wide binding targets in heads and bodies by ChIP-seq and show that they have distinct DNA targets in the two tissue contexts. Computational dissection of CLK/CYC context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, we show that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by OPA and SERPENT (SRP). They act synergistically with CLK/CYC in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, we validate that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock.ConclusionsOur results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression

HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature

HOT (highly occupied target) regions bound by many transcription factors are considered to be one of the most intriguing findings of the recent modENCODE reports, yet their functions have remained unclear. We tested 108 Drosophila melanogaster HOT regions in transgenic embryos with site-specifically integrated transcriptional reporters. In contrast to prior expectations, we found 102 (94%) to be active enhancers during embryogenesis and to display diverse spatial and temporal patterns, reminiscent of expression patterns for important developmental genes. Remarkably, HOT regions strongly activate nearby genes and are required for endogenous gene expression, as we show using bacterial artificial chromosome (BAC) transgenesis. HOT enhancers have a distinct cis-regulatory signature with enriched sequence motifs for the global activators Vielfaltig, also known as Zelda, and Trithorax-like, also known as GAGA. This signature allows the prediction of HOT versus control regions from the DNA sequence alone

Implementation and consequences of the EU directive 2006/24/EC in the context of internet access and e-mail

Zsfassg. in dt. SpracheDie EU Richtlinie 2006/24/EC vom 15.03.2006 schreibt den Betreibern öffentlicher Kommunikationsnetze vor, diverse Verkehrs- und Standort-Daten, die von ihnen erzeugt oder verarbeitet werden, auf Vorrat zu speichern, um sie auf Anfrage zur Ermittlung, Feststellung und Verfolgung von schweren Straftaten den Behörden zur Verfügung zu stellen. Diese Arbeit setzt sich mit den technischen und finanziellen Konsequenzen einer Implementierung der EU-Richtlinie für die Anbieter von Internetzugangs- und Internet-E-Mail Diensten auseinander. Der Ansatz, der hier gewählt wurde, ist der, dass ein Dienste-Anbieter nur die Teile der Richtlinie erfüllen muss, die für ihn zutreffen, er also auch entweder nur Internetzugangs-Anbieter oder nur E-Mail Dienstbetreiber sein kann. Um die Ergebnisse dieser Arbeit zu veranschaulichen, wurden die finanziellen Kosten und der benötigte Speicherplatz für einen fiktiven mittleren österreichischen Dienste-Anbieter geschätzt.The Data Retention directive 2006/24/EC of the European Parliament, released on 15.03.2006, requires the operators of publicly accessible electronic communication networks to store and provide traffic and location data generated or processed in their networks to serve the investigation, detection, and prosecution of serious crime.This thesis focuses on the technical and financial issues related to the implementation of the directive with respect to the guidelines for Internet access and Internet e-mail. Instead of each Internet Service Provider (ISP) having to implement the guidelines for both areas, the assumption made here is that service providers offering exclusively Internet access or Internet e-mail services may also be obliged to implement only one of the two areas. In order to illustrate the conclusions drawn in this thesis, cost estimations are made for a fictitious medium-sized Austrian service provider.8

Prevalence of Atrial Fibrillation and Antithrombotic Therapy in Hemodialysis Patients: Cross-Sectional Results of the Vienna InVestigation of AtriaL Fibrillation and Thromboembolism in Patients on HemoDIalysis (VIVALDI)

Background Atrial fibrillation (AF) adds significant risk of stroke and thromboembolism in patients on hemodialysis (HD). The aim of this study was to investigate the prevalence of AF in a population-based cohort of HD patients and practice patterns of antithrombotic therapy for stroke prevention in AF. Methods The Vienna InVestigation of AtriaL fibrillation and thromboembolism in patients on hemodialysis (VIVALDI), an ongoing prospective observational cohort study, investigates the prevalence of AF and the risk of thromboembolic events in HD patients in Vienna, Austria. We analyzed cross-sectional data of 626 patients (63.4% men, median age 66 years, approx. 73% of HD patients in Vienna), who provided informed consent. A structured interview with each patient was performed, recent and archived ECGs were viewed and medical histories were verified with electronic records. Results The overall prevalence of AF was 26.5% (166 patients, 71.1% men, median age 72 years) of which 57.8% had paroxysmal AF, 3.0% persistent AF, 32.5% permanent AF, and 6.6% of patients had newly diagnosed AF. The median CHA2DS2-VASc Score was 4 [25th-75th percentile 35]. In multivariable analysis, AF was independently associated with age (odds ratio: 1.05 per year increase, 95% confidence interval: 1.031.07), male sex (1.7, 1.12.6), history of venous thromboembolism (2.0, 1.13.6), congestive heart failure (1.7, 1.12.5), history of or active cancer (1.5, 1.02.4) and time on HD (1.08 per year on HD, 1.031.13). Antithrombotic treatment was applied in 84.4% of AF patients (anticoagulant agents in 29.5%, antiplatelet agents in 33.7%, and both in 21.1%). In AF patients, vitamin-K-antagonists were used more often than low-molecular-weight heparins (30.1% and 19.9%). Conclusions The prevalence of AF is high amongst HD patients and is associated with age, sex, and distinct comorbidities. Practice patterns of antithrombotic treatment indicate a lack of consensus for stroke prevention in HD patients with AF.(VLID)486840

Prevalence of atrial fibrillation in patients with increasing age and time on hemodialysis.

<p>Prevalence of atrial fibrillation in patients with increasing age and time on hemodialysis.</p

Factors associated with prevalence of atrial fibrillation Model 1 (N = 626).

<p>Factors associated with prevalence of atrial fibrillation Model 1 (N = 626).</p

Model 2 of factors associated with atrial fibrillation, excluding patients with a diagnosis of atrial fibrillation before occurrence of ESRD (N = 559).

<p>Model 2 of factors associated with atrial fibrillation, excluding patients with a diagnosis of atrial fibrillation before occurrence of ESRD (N = 559).</p

Characteristics of the study population at baseline.

<p>Characteristics of the study population at baseline.</p