Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region

Oxidized DNA bases, particularly 7,8-dihydro-8-oxoguanine (8-oxoG), are endogenously generated in cells, being a cause of carcinogenic mutations and possibly interfering with gene expression. We found that expression of an oxidatively damaged plasmid DNA is impaired after delivery into human host cells not only due to decreased retention in the transfected cells, but also due to selective silencing of the damaged reporter gene. To test whether the gene silencing was associated with a specific change of the chromatin structure, we determined the levels of histone modifications related to transcriptional activation (acetylated histones H3 and H4) or repression (methylated K9 and K27 of the histone H3, and histone H1) in the promoter region and in the downstream transcribed DNA. Acetylation of histone H4 was found to be specifically decreased by 25% in the proximal promoter region of the damaged gene, while minor quantitative changes in other tested chromatin components could not be proven as significant. Treatment with an inhibitor of histone deacetylases, trichostatin A, partially restored expression of the damaged DNA, suggesting a causal connection between the changes of histone acetylation and persistent gene repression. Based on these findings, we propose that silencing of the oxidatively damaged DNA may occur in a chromatin-mediated mechanism

Conducting interactive experiments online

Online labor markets provide new opportunities for behavioral research, but conducting economic experiments online raises important methodological challenges. This particularly holds for interactive designs. In this paper, we provide a methodological discussion of the similarities and differences between interactive experiments conducted in the laboratory and online. To this end, we conduct a repeated public goods experiment with and without punishment using samples from the laboratory and the online platform Amazon Mechanical Turk. We chose to replicate this experiment because it is long and logistically complex. It therefore provides a good case study for discussing the methodological and practical challenges of online interactive experimentation. We find that basic behavioral patterns of cooperation and punishment in the laboratory are replicable online. The most important challenge of online interactive experiments is participant dropout. We discuss measures for reducing dropout and show that, for our case study, dropouts are exogenous to the experiment. We conclude that data quality for interactive experiments via the Internet is adequate and reliable, making online interactive experimentation a potentially valuable complement to laboratory studies

Charakterisierung der Proteine CEP164 und ppdpf sowie deren Einordnung in den mitotischen Kontext

Eine regelgerechte bipolare Mitose und die fehlerfreie Aufteilung duplizierter DNA ist die Voraussetzung für die Entwicklung aller Lebewesen. Treten Fehler bei diesem grundsätzlichen Prozess auf, ist entweder der Zelltod oder die maligne Entartung der Zelle die Folge. Daher ist es von zentraler Bedeutung, die Vorgänge während der Zellteilung zu verstehen und die Funktion der an diesem Prozess beteiligten Proteine aufzudecken. Im Vorfeld dieser Arbeit wurden im Rahmen eines siRNA-Screens genomweit alle Proteine durch RNA-Interferenz depletiert und die Mitosen nach erfolgter RNAi phänotypisch untersucht. Ein besonderes Augenmerk lag dabei auf der Entwicklung multipolarer Spindeln durch Defekte in der Zentrosomenbündelung. Dadurch wurden unter anderem die Proteine CEP164 und ppdpf identifiziert. Da weder für CEP164 noch für ppdpf mitotische Funktionen bekannt sind, war es Ziel dieser Arbeit, die beiden Proteine eingehender zu charakterisieren und in den Kontext der Mitose einzuordnen. rnIm Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass CEP164 einer komplexen mitotischen Regulation unterliegt. Die in Interphase durchweg zentrosomale Lokalisation von CEP164 geht in der Mitose verloren. Es wird demonstriert, dass CEP164 während der Mitose unter anderem von CDK1 phosphoryliert wird und des Weiteren ubiquitinyliert wird. Als Interaktionspartner wurde das zentrosomale Protein Ninein identifiziert und demonstriert, dass sich CEP164 mit diesem in einem Komplex von ~2MDA befindet. Als weiterer Interaktionspartner wurde das Ninein-like-Protein ermittelt. Im Hinblick auf die Induktion multipolarer Mitosen wurde gezeigt, dass die Depletion von CEP164 nicht dafür verantwortlich ist. Die Induktion multipolarer Spindeln ist stattdessen darin begründet, dass durch die Transfektion einer siRNA gegen CEP164 auch ein für die Ausbildung der mitotischen Spindel elementares Protein, Ch-TOG, depletiert wird.rnIm Gegensatz dazu wurde im Rahmen dieser Arbeit bestätigt, dass das Protein ppdpf eine wichtige mitotische Funktion übernimmt. Zwar führt die Depletion von ppdpf nur zu einer sehr geringen Zunahme multipolarer Mitosen, allerdings steigt die Zahl aberranter Mitosen deutlich an, während die Spannung innerhalb mitotischer Spindeln abnimmt. Desweiteren konnte nachgewiesen werde, dass ppdpf-RNAi die Entwicklung von „lagging chromosomes“ und nachfolgend von Mikrokernen begünstigt. Es wurde gezeigt, dass ppdpf während der Mitose an Spindelmikrotubuli lokalisiert und spezifisch acetyliertes Tubulin bindet. Diese Interaktion hatte allerdings keinen Einfluss auf die Stabilität von Mikrotubuli während der Mitose. Das Protein ppdpf interagiert zudem mit dem Kinesin Eg5, wobei ppdpf-RNAi allerdings nicht zu einer Modulation der Aktivität von Eg5 zu führen scheint. Inwiefern diese Eigenschaften die Entwicklung von „lagging chromosomes“ begünstigen ist derzeit noch offen.The faithful segregation of chromosomes is elementary for the existence of life. Errors in this process either lead to cell death or may increase the risc of tumorigenic transformation. Hence, it is of utmost importance to delineate the functions of proteins during this fundamental process. In preparation of this work a genome-wide siRNA screen has been conducted with the aim of identifying proteins with an influence on centrosomal clustering. This work led to the identification of the proteins CEP164 and ppdpf as being neccessary for a regular mitosis. Since no mitotic function has so far been ascribed to either CEP164 or ppdpf, this work aims at characterizing these two proteins and elucidating their mitotic functions.rnHere it is demonstrated that CEP164 is subject to a complex mitotic regulation. The protein itself is extensively modified and the centrosomal localisation of CEP164 is lost at the onset of mitosis. The posttranslational modifications include phosphorylation by CDK1 and ubiquitinylation. In this work it has been shown that CEP164 interacts with another centrosomal protein, Ninein, and is part of a ~2MDa complex. Furthermore, CEP164 also interacts with the protein Ninein-like-Protein. Unfortunately, the induction of multipolar mitosis cannot be attributed to the depletion of CEP164. Instead it can be demonstrated that an off-target effect of the siRNA used strongly diminishes the levels of a crucial factor for bipolar spindle assembly, namely Ch-TOG.rnWith regard to ppdpf it is shown that although it does not seem to be essential for bipolar spindle assembly, ppdpf does have an important role for the accurate segregation of chromosomes during mitosis. Upon depletion of ppdpf the amount of lagging chromosomes and consequently, micronuclei increase, accompanied by a loss of spindletension. Depletion of ppdpf though, has no effect on microtubule-stability during mitosis. Furthermore, endogeneous ppdpf exclusively localizes to mitotic spindles during mitosis and an interaction with acetylated tubulin is demonstrated along with an interaction with the kinesin Eg5. How these functions lead to the development of lagging chromosomes upon depletion of ppdpf however remains obscure, but modulation of Eg5 activity can be excluded

Efficient contracting and fair play in a simple principal-agent experiment

We study behavior within a simple principal--agent experiment. Our design allows for a large class of linear contracts. Principals can offer any feasible combination of (negative) fixed wages and incentives in the form of return sharing. This great contractual flexibility allows us to study incentive compatibility simultaneously with issues of `fair sharing' and reciprocity, which were previously found to be important. We find a high degree of incentive-compatible behavior, but also `fair sharing' and reciprocity. In contrast to other incentive devices studied in the literature, the incentives are `reciprocity-compatible'. Principals recognize the agency problem and react accordingly

Efficient Contracting and Fair Play in a Simple Principal-Agent Experiment

We study behavior within a simple principal--agent experiment. Our design allows for a large class of linear contracts. Principals can offer any feasible combination of (negative) fixed wages and incentives in the form of return sharing. This great contractual flexibility allows us to study incentive compatibility simultaneously with issues of `fair sharing' and reciprocity, which were previously found to be important. We find a high degree of incentive-compatible behavior, but also `fair sharing' and reciprocity. In contrast to other incentive devices studied in the literature, the incentives are `reciprocity-compatible'. Principals recognize the agency problem and react accordingly.Principal-agent theory, contract theory, fair sharing, incentive contracts, reciprocity, experiments

Efficient Contracting and Fair Play in a Simple Principal-Agent Experiment

We study behavior within a simple principal-agent experiment. Our design allows for a large class of linear contracts. Principals can offer any feasible combination of (negative) fixed wages and incentives in the form of return sharing. This great contractual flexibility allows us to study incentive compatibility simultaneously with issues of 'fair sharing' and reciprocity, which were previously found to be important. We find a high degree of incentive-compatible behavior, but also 'fair sharing' and reciprocity. In contrast to other incentive devices studied in the literature, the incentives are 'reciprocity-compatible'. Principals recognize the agency problem and react accordingly. Copyright Kluwer Academic Publishers 2002principal-agent theory, contract theory, fair sharing, incentive contracts, reciprocity, experiments,

Influences of histone deacetylase inhibitors and resveratrol on DNA repair and chromatin compaction

Accessibility of DNA is a prerequisite for both DNA damage and repair. Therefore, the chromatin structure is expected to have major impact on both processes, with opposite consequences for the stability of the genome. To analyse the influence of chromatin compaction on the generation and repair of various types of DNA modifications, we modulated the global chromatin structure of AS52 Chinese hamster ovary cells and HeLa cells by treatment with either histone deacetylase inhibitors or resveratrol and measured the repair kinetics of (i) pyrimidine dimers induced by ultraviolet B, (ii) oxidised purines generated by photosensitisation and (iii) single-strand breaks induced by H2O2, using an alkaline elution technique. The decrease of chromatin compaction (detected as reduced DNA accessibility to DNase I) after treatment with trichostatin A or butyrate slightly increased the damage generation but had no significant effect on the global repair rates. In contrast, incubation of AS52 cells with resveratrol at concentrations that caused significant chromatin compaction and that had only moderate influence on cell proliferation gave rise to a strong decrease of the repair rates of all three types of DNA modifications. Similar, but less pronounced effects were observed in HeLa cells. The effects of resveratrol on the repair rates were not antagonised by the sirtuin inhibitor EX-527 or by an increase of the intracellular thiol levels