Isolation and identification of molecular partners of the proteins encoded by the Drosophila tumor suppressor gene lethal(2)tumorous imaginal discs

Ziel dieser Arbeit war es, die funktionelle Bedeutung des Drosophila melanogaster tumor suppressor Gens lethal(2)tumorous imaginal discs (l(2)tid) durch die Identifikation von molekularen Partnern der vom Gen kodierten Proteine zu etablieren. Mit dem Screening einer Expressionsbibliothek mittels des Hefe-Di-Hybrid-Systems wurde das Protein Patched (Ptc) als ein neues Tid-bindendes Protein identifiziert. Ptc ist ein Zentralregulator der Hedhehog-Signalkette. Diese ist in der Entwicklung konserviert und in manchen humanen Krebsarten verwickelt. Die Tid/Ptc-Interaktion wurde mittels unabhängigen biochemischen Methoden wie dem GST-pulldown-Test oder der Immunopräzipitation überprüft. Außerdem ergaben funktionelle Studien in tumorosen Imaginalscheiben einen möglichen inhibitorischen Effekt von Tid über die Hh Signaltransduktion.Im letzten Teil dieser Arbeit wurde die Interaktion zwischen Tid und dem E-APC-Protein (Adenomatous polyposis coli) bewiesen. Polakis und seine Gruppe zeigten durch Studien mit dem Hefe-Di-Hybrid-System und in vitro, dass das hTid mit dem APC-Protein interagiert. Um dies auch auf Drosophila-Ebene zu überprüfen, wurden Immunopräzipitation-Studien mit den Drosophila-Gegenstücken durchgeführt. Diese Studien zeigen zum ersten Mal eine direkte Interaktion beider Proteine in vivo.

Surface plasmon fluorescence spectroscopy and microscopy studies for biomolecular interaction studies

ABSTARCT Biotechnology has enabled the modification of agricultural materials in a very precise way. Crops have been modified through the insertion of new traits or the inhibition of existing gene functions, named Genetically Modified Organism (GMO), and resulted in improved tolerance of herbicide and/or increased resistance against pests, viruses and fungi. Commercial cultivation of GMO started in 1996 and increased rapidly in 2003 according to a recently released report by the International Service for the Acquisition of Agri-Biotech Applications (ISAAA), depicted continuing consumer resistance in Europe and other part of the world. Upon these developments, the European Union regulations mandated labeling of GMOs containing food and as a consequence, the labeling of GMO containing product in the case of exceeding the1% threshold of alien DNA is required. The aim of the study is to be able to detect and quantify the GMO from the mixture of natural food components. The surface plasmon resonance (SPR) technique combined with fluorescence was used for this purpose. During the presented studies, two key issues are addressed and tried to solve; what is the best strategy to design and built an interfacial architecture of a probe oligonucletide layer either on a two dimensional surface or on an array platform; and what is the best detection method allowing for a sensitive monitoring of the hybridisation events. The study includes two parts: first part includes characterization of different PNAs on a 2D planar surface by defining affinity constants using the very well established optical method “Surface Plasmon Fluorescence Spectroscopy”(SPFS) and on the array platform by “Surface Plasmon Fluorescence Microscopy” (SPFM), and at the end comparison of the sensitivity of these two techniques. The second part is composed of detection of alien DNA in food components by using DNA and PNA catcher probes on the array platform in real-time by SPFM.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Nukleation pflanzlicher Mikrotubuli: Expression relevanter Gene

Die wichtigsten Bestandteile des Cytoskeletts in pflanzlichen Zellen sind die Actinfilamente und die Mikrotubuli. Die Mikrotubuli spielen in der Organisation und der Morphogenese von pflanzlichen Zellen eine wichtige Rolle. Sie sind zusammen mit den Cellulosefibrillen an der Formgebung der Pflanzenzelle beteiligt. Sie bilden das Präprophaseband, das die Zellteilungsebene bestimmt und die Mitosespindel, die für die Trennung der Chromosomen sorgt, sowie den Phragmoplasten, der die Zellwand zwischen den Tochterzellen bildet. Weiterhin geben die Mikrotubuli durch Interaktion mit den Cellulose-Synthase-Komplexen die Richtung der Zellexpansion vor (GRANGER und CYR, 2001; LLOYD und CHAN, 2002; BASKIN, 2002). Die Mikrotubuli sind auch an der Stabilisierung der Zellform und an Transportprozessen beteiligt. Als Bestandteil der Mikrotubuli-organisierenden Zentren (MTOCs) wurde das γ-Tubulin identifiziert, das sehr wahrscheinlich an der Nukleation der Mikrotubuli beteiligt ist, indem es die Assemblierung der αβ-Tubulindimere zu Mikrotubuli einleitet. In tierischen Zellen ist durch intensive Forschung inzwischen relativ viel über die Funktion von γ-Tubulin, vor allem im Verlauf der Zellteilung bekannt, wie z. B. die Lokalisation in Centrosomen mit ihren paarweise angeordneten Centriolen, die die MTOCs darstellen. In pflanzlichen Zellen sind bisher nur wenige Funktionen des Proteins hinreichend geklärt. Die höheren Pflanzen besitzen keine Centriolen und keine Centrosomen. Über die Zellteilung hinaus gibt es kaum Anhaltspunkte über das Vorhandensein oder eventuelle Aufgaben von γ-Tubulin in expandierenden und voll expandierten Zellkulturen und Pflanzengeweben. In dieser Arbeit wurde die Expression über PCR und die Messung des Proteingehalts von cytoskelett-relevanten Proteinen in den Entwicklungsstadien der Zellsuspensionskultur (BY-2) und von Blattstadien der Tabakpflanze (SR1) von Nicotiana tabacum gemessen. Primäres Ziel war es eine Aussage zu erhalten, in welchem Ausmaß γ-Tubulin in expandierenden und voll expandierten Zellen noch exprimiert wird und ob bzw. wie eine Regulation (transkriptionell oder posttranskriptionell) des γ-Tubulins in der Pflanze stattfindet. Für den Nachweis des γ-Tubulins auf der Proteinebene wurde ein pflanzenspezifischer γ-Tubulin Antikörper zu entwickelt. Bei diesem Antikörper handelte es sich um einen polyklonalen Antikörper, der spezifisch gegen eine Sequenz in pflanzlichem γ-Tubulin gerichtet ist. Dabei zeigte der in der Arbeit entwickelte Antikörper gegen die pflanzliche JOSHI-Domäne spezifische Signale. Der erfolgte Nachweis von γ-Tubulin auf der Proteinebene und der Transkripte zeigte bis in die ältesten untersuchten Stadien der Zellsuspensionskultur (BY-2) und in Geweben der Blattstadien der Tabakpflanze (SR1) deutliche Signale für γ-Tubulin. Es war somit nicht nur in meristematisch aktiven Zellen und Geweben von Nicotiana tabacum, sondern auch in nichtmitotischen Zellen und Geweben vorhanden. Hierbei war über die Phasen der Zellteilung und der Zellformgebung hinweg auf beiden Ebenen eine parallele Entwicklung mit relativ konstanten starken Signalen zu beobachten. Nach dem Einstellen der Teilungsaktivität fiel der Gehalt an mRNA deutlich ab. Dabei nahm die Konzentration des Proteins im Vergleich zur mRNA zeitlich verzögert ab. Diese Ergebnisse bei der Zellsuspensionskultur (BY-2) und Tabakpflanze (SR1) gehen mit der möglichen Nukleationstätigkeit des Proteins konform. Es waren geringere aber doch deutlichen Signale bei Absterbenden Zellen der Zellkultur, bzw. bei expandierenden und voll expandierten und seneszenten Blättern der Tabakpflanze (SR1) nachzuweisen. Dies lässt die Folgerung zu, dass die nachgewiesene mRNA von γ-Tubulin nicht posttranskriptionell reguliert wird, sondern dass das γ-Tubulin auch eine wichtige Rolle außerhalb der Zellteilung in den postmitotischen Stadien, z. B. als organisierender Faktor bei der Umgestaltung oder Stabilisierung des Mikrotubuli-Cytoskeletts, spielt. Der γ-Tubulin-Gehalt in den Geweben der SR1-Pflanze zeigte über die Zellkultur hinaus, dass die Expression von α-Tubulin nach Einstellen der Teilungsaktivität kontinuierlich abnimmt. Dieses Ergebnis legt die Vermutung nahe, dass γ-Tubulin in älteren Blattgeweben zusätzliche Aufgaben übernehmen könnte, die nicht auf eine gleichzeitige Expression von α-Tubulin angewiesen sind. So kann beispielsweise eine Beteiligung von γ-Tubulin an der Stabilisierung der Mikrotubuli, und damit einhergehend eine Abnahme der dynamischen Instabilität dieser Filamente, eine denkbare Funktion des Proteins in expandierendem und voll expandiertem Gewebe sein. Die Aufgaben von γ-Tubulin in sehr altem Gewebe mit deutlichen Anzeichen der Seneszenz können allerdings nach dem derzeitigen Stand der Forschung nicht eindeutig beantwortet werden und bedürfen weitergehenden Untersuchungen, da dadurch ein die Komplexität und die Dynamik des pflanzlichen Cytoskeletts geklärt werden kann.

Oligomerisation, localisation and interaction of the sensor histidine kinases DcuS and CitA in Escherichia coli

The two-component system DcuSR of Escherichia coli regulates gene expression of anaerobic fumarate respiration and aerobic C4-dicarboxylate uptake. C4-dicarboxylates and citrate are perceived by the periplasmic domain of the membrane-integral sensor histidine kinase DcuS. The signal is transduced across the membrane by phosphorylation of DcuS and of the response regulator DcuR, resulting in activation of DcuR and transcription of the target genes.rnIn this work, the oligomerisation of full-length DcuS was studied in vivo and in vitro. DcuS was genetically fused to derivatives of the green fluorescent protein (GFP), enabling fluorescence resonance energy transfer (FRET) measurements to detect protein-protein interactions in vivo. FRET measurements were also performed with purified His6-DcuS after labelling with fluorescent dyes and reconstitution into liposomes to study oligomerisation of DcuS in vitro. In vitro and in vivo fluorescence resonance energy transfer showed the presence of oligomeric DcuS in the membrane, which was independent of the presence of effector. Chemical crosslinking experiments allowed clear-cut evaluation of the oligomeric state of DcuS. The results showed that detergent-solubilised His6-DcuS was mainly monomeric and demonstrated the presence of tetrameric DcuS in proteoliposomes and in bacterial membranes.rnThe sensor histidine kinase CitA is part of the two-component system CitAB of E. coli, which is structurally related to DcuSR. CitAB regulates gene expression of citrate fermentation in response to external citrate. The sensor kinases DcuS and CitA were fused with an enhanced variant of the yellow fluorescent protein (YFP) and expressed in E. coli under the control of an arabinose-inducible promoter. The subcellular localisation of DcuS-YFP and CitA-YFP within the cell membrane was studied by means of confocal laser fluorescence microscopy. Both fusion proteins were found to accumulate at the cell poles. The polar accumulation was slightly increased in the presence of the stimulus fumarate or citrate, respectively, but independent of the expression level of the fusion proteins. Cell fractionation demonstrated that polar accumulation was not related to inclusion bodies formation. The degree of polar localisation of DcuS-YFP was similar to that of the well-characterised methyl-accepting chemotaxis proteins (MCPs), but independent of their presence. To enable further investigations on the function of the polar localisation of DcuS under physiological conditions, the sensor kinase was genetically fused to the flavin-based fluorescent protein Bs2 which shows fluorescence under aerobic and anaerobic conditions. The resulting dcuS-bs2 gene fusion was inserted into the chromosome of various E. coli strains.rnFurthermore, a protein-protein interaction between the related sensor histidine kinases DcuS and CitA, regulating common metabolic pathways, was detected via expression studies under anaerobic conditions in the presence of citrate and by in vivo FRET measurements.

Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.

Kaiso und CTCF regulieren geprägte Gene in cis und in trans

Geprägte Gene besitzen die Besonderheit, dass sie jeweils nur von einem Allel exprimiert werden und in der Regel in Imprinting Clustern (ICs) im Genom vorliegen. Bei der Regulation in solchen ICs spielen differentiell methylierte Imprinting Kontrollregionen (ICRs) und dort stattfindende Proteinbindungen eine wichtige Rolle. Die essentielle Bedeutung der CTCF-Bindung an die ICR1 in 11p15.5 für die Expressionsregulation der geprägten Gene H19 und IGF2 ist bereits bekannt. In der vorliegenden Arbeit sollte die Bindung von Kaiso an die unmethylierte ICR1 bei humanen Zellen mit maternaler uniparentaler Disomie von 11p15 (upd(11p15)mat) nachgewiesen und die genaue Bindungsverteilung von Kaiso und CTCF in den B-Repeats der Kontrollregion bestimmt werden. Cis-regulatorische und chromosomenübergreifende transkriptionelle Effekte der ICR1-Proteinbindungen sollten dann durch qPCR-Analysen geprägter Gene bei Zellen mit maternaler und paternaler upd(11p15) und nach siRNA-basierter Herunterregulation der beiden Proteine in Zellen mit upd(11p15)mat analysiert werden. In der vorliegenden Arbeit konnte erstmals gezeigt werden, dass Kaiso an die unmethylierte ICR1 bindet. Dabei kann zumindest von einer Bindestellennutzung in der distalen ICR1-Hälfte ausgegangen werden. Für CTCF hingegen wurde eine Nutzung aller analysierten Repeats in beiden ICR1-Hälften gefunden. In der maternalen bzw. paternalen upd(11p15) entspricht die Expression der 11p15.5-Gene IGF2, H19, CDKN1C und KCNQ1OT1 dem jeweiligen Disomie-Status. Von den nicht auf Chromosom 11 gelegenen geprägten Genen zeigen MEST und PLAGL1 bei Zellen mit upd(11p15)pat sowie PEG3 und GRB10 bei der upd(11p15)mat eine stärkere Expression. Ein CTCF-knockdown in Zellen mit upd(11p15)mat führt zur IGF2-Expressionssteigerung. Dies tritt in noch stärkerem Maße beim knockdown von Kaiso auf, wobei hier zusätzlich eine gesteigerte Expression von H19 vorliegt. Des Weiteren findet man beim CTCF-knockdown einen MEST-Expressionsanstieg und beim Kaiso-knockdown gesteigerte Expressionen der Gene PEG3, GRB10 und PLAGL1. Damit lassen sich sowohl eigenständige cis-regulatorische Effekte der ICR1-Bindung beider Proteine auf geprägte Gene des IC1 als auch chromosomenübergreifende Effekte erkennen. Vor allem die starken H19-Expressionsanstiege beim Kaiso-knockdown treten korrelierend mit Veränderungen von geprägten Genen anderer Chromosomen auf. Damit unterstützen die Daten die Theorie, dass die Expressionsregulation geprägter Gene koordiniert in einer Art Netzwerk stattfinden könnte und dabei bestimmte Faktoren wie H19 und PLAGL1 eine übergeordnete Regulatorfunktion besitzen, wie es in Vergangenheit in der Maus beschrieben wurde. Die Expressionsanalysen von PLAGL1 und MEST deuten darüber hinaus durch ihre tendenziell übereinstimmenden Werte bei der paternalen upd mit hypermethylierter ICR1 und den knockdowns auf die Existenz von Chromatin-Interaktionen zwischen der ICR1 und Abschnitten auf den Chromosomen 6 und 7 hin, ggf. mit einem entsprechenden lokalen Effekt der Proteine in diesen Loci. Proteinbindungen an die maternale ICR1 scheinen damit sowohl cis-regulatorisch die Transkription der geprägten Gene IGF2 und H19 zu beeinflussen als auch durch die H19-Expression ein funktionelles Netzwerk geprägter Gene als trans-Faktor zu regulieren und für Interaktionen zwischen verschiedenen Chromosomen mit transkriptionsregulierender Wirkung verantwortlich zu sein.

Chlamydiae host cell interaction and immune evasion strategies

Chlamydiae are obligate intracellular bacteria with a strong global prevalence. They cause infections of the eye, lung and the genital tract and can either replicate in inclusion compartments or persist inside their host cell. In this thesis we focused on two aspects of chlamydiae infection. We hypothesize that transcription factor AP-1 is crucial for a replicative chlamydiae infection in epithelial cells. In addition we suggest that chlamydiae hide inside apoptotic blebs for a silent uptake by macrophages as immune evasion strategy.rnFocusing on AP-1, we could demonstrate that during Chlamydia pneumoniae infection, protein expression and phosphorylation of the AP-1 family member c-Jun significantly increased in a time and dose dependent manner. A siRNA knockdown of c-Jun in HEp-2 cells reduced chlamydial load, resulting in smaller inclusions and a significant lower chlamydial recovery. Furthermore, inhibition of the c-Jun containing AP-1 complexes, using Tanshinone IIA, changed the replicative infection into a persistent phenotype, characterized by (i) smaller, aberrant inclusions, (ii) a strong decrease in chlamydial load, as well as by (iii) its reversibility after removal of Tanshinone IIA. As chlamydiae are energy parasites, we investigated whether Tanshinone IIA interferes with energy/metabolism related processes. rnA role for autophagy or gene expression of glut-1 and c-jun in persistence could not be determined. However we could demonstrate Tanshinone IIA treatment to be accompanied by a significant decrease of ATP levels, probably causing a chlamydiae persistent phenotype.rnRegarding the chlamydial interaction with human primary cells we characterized infection of different chlamydiae species in either pro-inflammatory (type I) or anti-inflammatory (type II) human monocyte derived macrophages (hMDM). We found both phenotypes to be susceptible to chlamydiae infection. Furthermore, we observed that upon Chlamydia trachomatis and GFP-expressing Chlamydia trachomatis infection more hMDM type II were infected. However the chlamydial load was higher in hMDM type I and correspondingly, more replicative-like inclusions were found in this phenotype. Next, we focused on the chlamydial transfer using a combination of high speed live cell imaging and GFP-expressing Chlamydia trachomatis for optimal visualization. Thereby, we could successfully visualize the formation of apoptotic, chlamydiae-containing blebs and the interaction of hMDM with these blebs. Moreover, we observed the development of a replicative infection in hMDM. rnIn conclusion, we demonstrated a crucial role of AP-1 for C. pneumoniae development and preliminary time lapse data suggest that chlamydiae can be transferred to hMDMs via apoptotic blebs. In all, these data may contribute to a better understanding of chlamydial infection processes in humans.rn

Regulation von "Silent mating type information regulation 2 homolog Type" 1 (SIRT1) durch die nach DNA-Schaden induzierte Kinase "Homeodomain-interaction kinase" (HIPK2)

"Silent mating type information regulation 2 Type" 1 (SIRT1), das humane Homolog der NAD+-abhängigen Histondeacetylase Sir2 aus Hefe, besitzt Schlüsselfunktionen in der Regulation des Metabolismus, der Zellalterung und Apoptose. Letztere wird vor allem durch die Deacetylierung von p53 an Lys382 und der dadurch verringerten Transkription proapoptotischer Zielgene vermittelt. Im Rahmen der vorliegenden Arbeit wurde die SIRT1 Regulation im Zusammenhang mit der DNA-Schadensantwort untersucht.rnIn der Apoptoseregulation übernimmt die Serin/Threonin-Kinase "Homeodomain interacting protein kinase" 2 (HIPK2) eine zentrale Rolle und daher wurde die SIRT1 Modifikation und Regulation durch HIPK2 betrachtet. Durch Phosphorylierung des Tumorsuppressorproteins p53 an Ser46 aktiviert HIPK2 das Zielprotein und induziert die Transkription proapoptotischer Zielgene von p53. Es wurde beschrieben, dass HIPK2 nach DNA-Schädigung über einen bisher unbekannten Mechnismus die Acetylierung von p53 potenzieren kann.rnIn der vorliegenden Arbeit konnte gezeigt werden, dass SIRT1 von HIPK2 in vitro und in Zellen an Serin 27 und 682 phosphoryliert wird. Weiterhin ist die Interaktion von SIRT1 mit HIPK2 sowie die SIRT1 Phosphorylierung an Serin 682 durch DNA-schädigende Adriamycinbehandlung erhöht. Es gibt Hinweise, dass HIPK2 die Expression von SIRT1 reguliert, da HIPK2 RNA-Interferenz zur Erniedrigung der SIRT1 Protein- und mRNA-Mengen führt.rnEin weiterer interessanter Aspekt liegt in der Beobachtung, dass Ko-Expression von PML-IV, welches SIRT1 sowie HIPK2 in PML-Kernkörper rekrutiert, die SIRT1 Phosphorylierung an Serin 682 verstärkt. Phosphorylierung von SIRT1 an Serin 682 interferiert wiederum mit der SUMO-1 Modifikation, welche für die Lokalisation in PML-Kernkörpen wichtig ist.rnBemerkenswerterweise reduziert die DNA-schadendsinduzierte SIRT1 Phosphorylierung die Bindung des SIRT1 Ko-Aktivators AROS, beeinflusst aber nicht diejenige des Inhibitors DBC1. Dies führt zur Reduktion der enzymatischen Aktivität von SIRT1 und der darausfolgenden weniger effizienten Deacetylierung des Zielproteins p53.rnDurch die von mir in der vorliegenden Promotionsarbeit erzielten Ergebnisse konnte ein neuer molekularer Mechanismus entschlüsselt werden, welcher die durch HIPK2 modulierte Acetylierung von p53 und die daran anschließende Induktion der Apoptose beschreibt.rnHIPK2-vermittelte SIRT1 Phosphorylierung resultiert in einer verminderten Deacetylasefunktion von SIRT1 und führt so zu einer verstärkten acetylierungsinduzierten Expression proapoptotischer p53 Zielgene.