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While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment.
Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells.
Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade.
We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation.
Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse.
The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H\(_2\)O\(_2\) or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
The Gram-negative, spiral-shaped, microaerophilic bacterium Helicobacter pylori is the causative agent of various disorders of the upper gastrointestinal tract, such as chronic superficial gastritis, chronic active gastritis, peptic ulceration and adenocarcinoma. Although many of the bacterial factors associated with disease development have been analysed in some detail in the recent years, very few studies have focused so far on the mechanisms that regulate expression of these factors at the molecular level. In an attempt to obtain an overview of the basic mechanisms of virulence gene expression in H. pylori, three important virulence factors of this pathogen, representative of different pathogenic mechanisms and different phases of the infectious process, are investigated in detail in the present thesis regarding their transcriptional regulation. As an essential factor for the early phase of infection, including the colonisation of the gastric mucosa, the flagella are analysed; the chaperones including the putative adhesion factors GroEL and DnaK are investigated as representatives of the phase of adherence to the gastric epithelium and persistence in the mucus layer; and finally the cytotoxin associated antigen CagA is analysed as representative of the cag pathogenicity island, which is supposed to account for the phenomena of chronic inflammation and tissue damage observed in the later phases of infection. RNA analyses and in vitro transcription demonstrate that a single promoter regulates expression of cagA, while two promoters are responsible for expression of the upstream divergently transcribed cagB gene. All three promoters are shown to be recognised by RNA polymerase containing the vegetative sigma factor sigma 80. Promoter deletion analyses establish that full activation of the cagA promoter requires sequences up to -70 and binding of the C-terminal portion of the alpha subunit of RNA polymerase to an UP-like element located between -40 and -60, while full activation of the major cagB promoter requires sequences upstream of -96 which overlap with the cagA promoter. These data suggest that the promoters of the pathogenicity island represent a class of minimum promoters, that ensure a basic level of transcription, while full activation requires regulatory elements or structural DNA binding proteins that provide a suitable DNA context. Regarding flagellar biosynthesis, a master transcriptional factor is identified that regulates expression of a series of flagellar basal body and hook genes in concert with the alternative sigma factor sigma 54. Evidence is provided that this regulator, designated FlgR (for flagellar regulatory protein), is necessary for motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR is shown to act as a repressor of transcription of the sigma 28-regulated promoter of the flaA gene, while changes in DNA topology are shown to affect transcription of the sigma 54-regulated flaB promoter. These data indicate that the regulatory network that governs flagellar gene expression in H. pylori shows similarities to the systems of both Salmonella spp. and Caulobacter crescentus. In contrast to the flagellar genes which are regulated by three different sigma factors, the three operons encoding the major chaperones of H. pylori are shown to be transcribed by RNA polymerase containing the vegetative sigma factor sigma 80. Expression of these operons is shown to be regulated negatively by the transcriptional repressor HspR, a homologue of a repressor protein of Streptomyces spp., known to be involved in negative regulation of heat shock genes. In vitro studies with purified recombinant HspR establish that the protein represses transcription by binding to large DNA regions centered around the transcription initiation site in the case of one promoter, and around -85 and -120 in the case of the the other two promoters. In contrast to the situation in Streptomyces, where transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible with thermal stimuli. Transcription of two of the three chaperone encoding operons is induced by osmotic shock, while transcription of the third operon, although HspR-dependent, is not affected by salt treatment. Taken together, the analyses carried out indicate that H. pylori has reduced its repertoire of specific regulatory proteins to a basic level that may ensure coordinate regulation of those factors that are necessary during the initial phase of infection including the passage through the gastric lumen and the colonisation of the gastric mucosa. The importance of DNA topology and/or context for transcription of many virulence gene promoters may on the other hand indicate, that a sophisticated global regulatory network is present in H. pylori, which influences transcription of specific subsets of virulence genes in response to changes in the microenvironment.
Background: Gene function analysis of the obligate intracellular bacterium Chlamydia pneumoniae is hampered by the facts that this organism is inaccessible to genetic manipulations and not cultivable outside the host. The genomes of several strains have been sequenced; however, very little information is available on the gene structure and transcriptome of C. pneumoniae. Results: Using a differential RNA-sequencing approach with specific enrichment of primary transcripts, we defined the transcriptome of purified elementary bodies and reticulate bodies of C. pneumoniae strain CWL-029; 565 transcriptional start sites of annotated genes and novel transcripts were mapped. Analysis of adjacent genes for cotranscription revealed 246 polycistronic transcripts. In total, a distinct transcription start site or an affiliation to an operon could be assigned to 862 out of 1,074 annotated protein coding genes. Semi-quantitative analysis of mapped cDNA reads revealed significant differences for 288 genes in the RNA levels of genes isolated from elementary bodies and reticulate bodies. We have identified and in part confirmed 75 novel putative non-coding RNAs. The detailed map of transcription start sites at single nucleotide resolution allowed for the first time a comprehensive and saturating analysis of promoter consensus sequences in Chlamydia. Conclusions: The precise transcriptional landscape as a complement to the genome sequence will provide new insights into the organization, control and function of genes. Novel non-coding RNAs and identified common promoter motifs will help to understand gene regulation of this important human pathogen.
Amphibians evolved in the Devonian period about 400 Mya and represent a transition step in tetrapod evolution. Among amphibians, high-throughput sequencing data are very limited for Caudata, due to their largest genome sizes among terrestrial vertebrates. In this paper we present the transcriptome from the fire bellied newt Cynops orientalis. Data here presented display a high level of completeness, comparable to the fully sequenced genomes available from other amphibians. Moreover, this work focused on genes involved in gametogenesis and sexual development. Surprisingly, the gsdf gene was identified for the first time in a tetrapod species, so far known only from bony fish and basal sarcopterygians. Our analysis failed to isolate fgf24 and foxl3, supporting the possible loss of both genes in the common ancestor of Rhipidistians. In Cynops, the expression analysis of genes described to be sex-related in vertebrates singled out an expected functional role for some genes, while others displayed an unforeseen behavior, confirming the high variability of the sex-related pathway in vertebrates.
In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from mature Xenopus laevis oocytes were manually fractioned into nucleo· plasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60- 90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the mature Xenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41 X 10- 16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270 X 10- 15 g/fJ-3) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules. Additionally, the results of the chemical analyses as well as of the 3H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.
1 n order to review the contradictory statements on the ultrast ructure of the nuclear envelope, a study was undertaken combining section and negat ive stai ning electron microscopy on manually isolated oocyte nuclei and nuclear envelopes from six amphibian species including Anura as well as Urodela. The a ppeara nce of the negatively stained iso lated nuclear envelopes is described in deta il and the dependence on the preparation co nditions used is emphas ized . Pore complex structures such as pore perimeter, central granule, an nul ar components, interna l fibrils, and annu lus-attached fibrils could be identified by both techniques, negat ive staining and sect ions. Comparative studies show that no marked diffe rences ex ist in the structural data of the nuclear envelope among the investigated amphibians and the significance of the structural components is discussed. A model of the nuclea r pore complex based on the findings of the present investigation is prese nted.