cis-Regulation des Drosophila-melanogaster-Gens optomotor-blind

Die zeitliche und räumliche Expression von Genen trägt zu einem entscheidenden Ausmaß zu der Entwicklung eines Organismus bei. Unter vielen Faktoren spielt dabei die transkriptionelle Regulation eine wichtige Rolle. Diese basiert auf Anwesenheit und Binden von regulatorischen Proteinen an cis-regulatorischen Sequenzen (CRMs) und deren Einfluss auf die Transkriptionsmaschinerie am Promotor. Veränderungen der CRMs können zu Veränderungen der Genexpression führen, und somit einen Beitrag zur morphologischen Evolution leisten. rnIn dieser Arbeit wurde die transkriptionelle Regulation des Drosophila melanogaster Gens optomotor-blind insbesondere in den pupalen Tergiten untersucht. In einem Enhancer-Reporter screen wurde eine regulatorische Region in Intron IV, die Reportergen-Expression in den pupalen Tergiten treibt, identifiziert. Große Teile dieser Region (ombTU10 und ombTU11) trieben Reportergen-Expression in einem omb-ähnlichen Muster. Eine weitere Region (ombTU12) trieb Expression in einem für Hh-Zielgene typischen Expressionsmuster. Für ombTU12 konnte eine Hh-Abhängigkeit nachgewiesen werden. Die für Hh-Zielgene typische Enhanceraktivität konnte in dem Subfragment ombTU12Amin lokalisiert werden, welches zwei konservierte Bindestellen des Effektors der Hh-Signaltransduktionskaskase, Cubitus interruptus (Ci), enthält. Eine deutliche Abhängigkeit der Expression dieses Fragments von den Ci-Bindestellen konnte bisher aber noch nicht nachgewiesen werden.rnDeletionen verschiedener Bereiche dieser Tergitenenhancer-Region aus dem endogenen Gen sollten Aufschluss über deren Notwendigkeit in der Regulation von omb geben. Die Deletion des Fragments ombTU10 (ΔombTU10-2) führte zu einer Variabilität in der Pigmentierung der Abdominalsegmente A5 und A6 der Weibchen. Eine Deletion von Teilen des hh-responsiven Fragments ombTU12 (ΔombTU12A) zeigte keinen abdominalen Phänotyp. Dies deutet auf eine redundante Wirkung der Fragmente untereinander, oder mit einem weiteren bisher nicht identifizierten Tergitenenhancer im omb-Locus hin.rnFragmente, die in den pupalen Tergiten Reportergen-Expression trieben, waren zum Teil auch in Imaginalscheiben von Larven aktiv. Desweiteren wurde gezeigt, dass Fragmente, die in Isolation Reportergen-Expression trieben, als Fusionskonstrukt mit benachbarten genomischen Sequenzen keine Expression zeigten und somit im genomischen Kontext inaktiv sein können. Demzufolge sind nicht nur Aktivator- sondern auch Repressorregionen für die korrekte Expression eines Gens von Bedeutung.rnDie Analyse von omb Enhancer-Trap Insertionen zeigte, dass von drei untersuchten Typen (PlacW, PGalW und PGawB) nur Insertionen vom letzteren in den pupalen Tergiten aktiv waren. Von vier PGawB Insertionen waren nur drei aktiv. Es ist denkbar, dass die Orientierung der inaktiven Insertion für die mangelnde Responsivität verantwortlich ist.rn

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.

The role of microglia in neurodegeneration and endogenous repair during experimental autoimmune encephalomyelitis

Inflammation-mediated neurodegeneration occurs in the acute and the chronic/progressive phases of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Classically-activated microglia (M1) are key players mediating this process through secretion of soluble factors including nitric oxide (NO) and tumor necrosis factor (TNF). Here, galectin-1, an endogenous glycan-binding protein, was identified as a pivotal regulatory mechanism that limits M1 microglia activation and neurodegeneration, by targeting the activation of p38MAPK- and CREB-dependent pathways and hierarchically controlling downstream pro-inflammatory mediators such as iNOS, TNF and CCL2. Galectin-1 is highly expressed in the acute phase of EAE and its targeted deletion results in pronounced inflammation-induced neurodegeneration. These findings identify an essential role of galectin-1-glycan lattices in tempering microglia activation, brain inflammation and neurodegeneration with critical therapeutic implications in relapsing-remitting and secondary progressive MS.rnMicroglia with distinct phenotypes are implicated in neurotoxicity, neuroprotection, and in modulation of endogenous repair by NSCs. However the precise molecular mechanisms underlying this diversity in fuction are still unknown. rnUsing a model of EAE, transcriptional profiling of isolated SVZ microglia from the acute and chronic disease phases of EAE was performed. The results from this study suggest that microglia exhibit disease phase specific gene expression signatures, that correspond to unique GO functions and genomic networks. These data demonstrate for the first time, distinct transcriptional networks of microglia activation in vivo, that support their role as mediators of injury or repair.

Regulatory T cell-mediated suppression of Th9 cell development and effector function

T helper (Th) 9 cells are an important subpopulation of the CD4+ T helper cells. Due to their ability to secrete Interleukin-(IL-)9, Th9 cells essentially contribute to the expulsion of parasitic helminths from the intestinal tract but they play also an immunopathological role in the course of asthma. Recently, a beneficial function of Th9 cells in anti-tumor immune responses was published. In a murine melanoma tumor model Th9 cells were shown to enhance the anti-melanoma immune response via the recruitment of CD8+ T cells, dendritic cells and mast cells. In contrast to Th9 effector cells regulatory T cells (Tregs) are able to control an immune response with the aid of different suppressive mechanisms. Based on their ability to suppress an immune response Tregs are believed to be beneficial in asthma by diminishing excessive allergic reactions. However, concerning cancer they can have a detrimental function because Tregs inhibit an effective anti-tumor immune reaction. Thus, the analysis of Th9 suppression by Tregs is of central importance concerning the development of therapeutic strategies for the treatment of cancer and allergic diseases and was therefore the main objective of this PhD thesis.rnIn general it could be demonstrated that the development of Th9 cells can be inhibited by Tregs in vitro. The production of the lineage-specific cytokine IL-9 by developing Th9 cells was completely suppressed at a Treg/Th9 ratio of 1:1 on the transcriptional (qRT-PCR) as well as on the translational level (ELISA). In contrast, the expression of IRF4 that was found to strongly promote Th9 development was not reduced in the presence of Tregs, suggesting that IRF4 requires additional transcription factors to induce the differentiation of Th9 cells. In order to identify such factors, which regulate Th9 development and therefore represent potential targets for Treg-mediated suppressive mechanisms, a transcriptome analysis using “next-generation sequencing” was performed. The expression of some genes which were found to be up- or downregulated in Th9 cells in the presence of Tregs was validated with qRT-PCR. Time limitations prevented a detailed functional analysis of these candidate genes. Nevertheless, the analysis of the suppressive mechanisms revealed that Tregs probably suppress Th9 cells via the increase of the intracellular cAMP concentration. In contrast, IL-9 production by differentiated Th9 cells was only marginally affected by Tregs in vitro and in vivo analysis (asthma, melanoma model). Hence, Tregs represent very effective inhibitors of Th9 development whereas they have only a minimal suppressive influence on differentiated Th9 cells.rn