Browsing by Subject "Epigenetik"

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Beiträge zur Verbesserung der Analytik von Mutationen im Protoonkogen Kirsten-ras und epigenetische Untersuchungen zur Eignung von DUSP9/MKP4 als CIMP-Marker
(2012) Jenner, Stefan; Preiss, Anette
The present study extends over two different fields of applications, which contribute to improvements for the analysis of colorectal cancers. First, a ligase-based method was developed which allows a quick, inexpensive, specific and highly sensitive detection of point mutations in the mutation hot spot codon12 and codon13 of the K-ras gene. The establishment and validation of the technique was performed on clinical samples, which were present as FFPE tissues and gone through routine diagnostics using conventional molecular biological techniques (microarray analysis, Sanger Sequencing) to determine the K-ras mutation status. In addition, a comparison between the new developed technology and the conventional technologies should be performed. The evaluation of the gLCR approach was done by ABI310er capillary electrophoresis. Among the tested LCR variants the gLCR-monoplex had been the most robust, specific and sensitive technique. The presence of very weak mutations in the samples had been successfully confirmed by gLCR-monoplex, whereas several conventional techniques had to be applied together to detect the mutations unambiguously. The signal strengths for all tested samples were high, coming along with a low standard deviation. The superiority of gLCR-monoplex over the conventional techniques had been further highlighted impressively by performing a comparison of methods on a dilution series. While the mutation detection using Sanger Sequencing or microarray analysis had been successful only up to the 1:10-dilution, or 1:100-dilution, it was possible to detect the K-ras mutation by gLCR-technique up to the 1:1 million-dilution. In routine diagnostics a single monoplex reaction for each possible mutation has to be performed. Therefore the monoplex technique is only suitable as a confirmatory test of weak or doubtful mutation screening results, which had been previously indicated by conventional techniques. A multiplex approach would be desirable to use the gLCR technology as a main detection technique in routine diagnostics. Therefore a single discriminating base at the mutation-specific and color-labeled oligo probe appears to be insufficient. In future studies an additional mismatch should be integrated at position (-3), in relation to the 3'-end of the dye-labeled and mutation-specific oligo probe. In this way it could come to a significant reduction of false-positive signals, thereby gLCR technology could possibly be used as a multiplex approach. In the second part of the work the methylation status of the DUSP9/MKP4 promoter region had been evaluated. By methylation, the accessibility of the promoter, and thus the transcriptional activation of a gene can be reduced. The promoter regions of tumor suppressors are frequently strong methylated in colorectal cancer (CRC). The methylation is resulting in an increased tumorigenesis and cannot be observed in the corresponding normal tissues. This alteration is called CIMP (CpG-island-methylator-phenotype) and is an independent tumor phenotype, which is linked to other clinical aspects. Involved genes are classified as CIMP markers. The DUSP9/MKP4 gene product is a potent tumor suppressor, but it´s suitability as a marker for CIMP in CRC has not been studied so far. In this study, the degree of methylation of 79 colorectal cancer FFPE tissue samples and 22 corresponding normal FFPE tissues was determined quantitatively. A broad variation of methylation strength has been observed in the examined tumor tissues, ranging from nearly unmethylated to nearly completely methylated. Only minor differences between tumor and normal tissues had been detected for the 11 DNA samples with the lowest methylation strength. On the other side, there was a significant correlation with CRC for the 11 strongest methylated DNA samples. About 80% of the DNA from normal tissue showed clearly weaker methylation, leading to the conclusion, that an aberrant DNA methylation status is present in these tumor tissues. Additionally, 9 out of 11 strong methylated DNA samples showed CIMP criteria, which were completely missing at the weakly methylated DNA samples. This work represents, as far as published, the first study that reveals a difference in the methylation pattern of the DUSP9/MKP4 promoter from human colorectal carcinoma and their corresponding normal tissues. Strong methylation could be associated with all tested CIMP criteria. This relationship needs to be confirmed in further studies on a larger sample collective. In addition, the investigations should include the detection of microsatellite instabilities, as an additional CIMP-criterion, and a functional protein detection method should be established.
Interplay between nutrition, senescence and cytosine methylation in Arabidopsis thaliana
(2023) Vatov, Emil; Ludewig, Uwe
In monocarpic plants, senescence is the last stage of leaf development and usually leads to the death of the organism. Systematic degradation of leaf components provides nutrients for the newly developing flowers and seeds. The physiology and transcriptional changes that occur in A. thaliana during this process are very well documented. However, the involvement of epigenic mechanisms remains to be established. In this study, the role of cytosine methylation in the regulation of monocarpic leaf senescence was examined in A. thaliana. Hypomethylated ddc (drm1/2 cmt3) and hypermethylated ros1 mutants showed consistent senescence-specific phenotypes. Disrupted de-novo methylation resulted in delayed, while disrupted demethylation resulted in earlier flowering and appearance of first symptoms of senescence. Both genotypes executed the senescence program faster than Col-0, with lower leaf:seed and higher C:N ratios. During nitrogen, or phosphorus withdrawal and resupply, nutrient remobilization was not inhibited in the two mutants. However, the plant’s response in terms of changes in shoot and root growth was delayed, or non existent. Furthermore, the impact of N withdrawal on delay of the flowering time was inhibited in the two mutants. These results support involvement of cytosine methylation in stress response signaling and downstream effects on organ development and flowering times. The stress response and senescence specific phenotypes of ddc could be partially due to disrupted WRKY signaling, as loss of methylation in W-box binding sites was prevalent, specifically near the transcription start sites of ORFs, and WRKY18, 25 and 53 appeared to be sensitive towards cytosine methylation. Overall decrease in cytosine methylation levels was observed, as early as the opening of the first flowers, together with a decrease in chlorophyll concentrations and an increase in H2O2 and glucose levels in the wild type Col-0. Inhibition in maintenance methylation in the early stages of reproductive growth is consistent with these observations. A complex interaction between four cytokinins was present as early as flower induction, followed by a mass turnover of bound auxin (IAA) at flower opening, that resulted in near doubling of free IAA at seed development. Plant defense responses were induced thereafter, as an increase in salicylic acid (SA) and camalexin occurred, followed by an increase in jasmonic acid (JA) and abscisic acid (ABA). Active RNA-dependent DNA methylation (RdDM) was indicated by a moderate overrepresentation of hypermethylated CHG and CHH loci, together with partial recovery of total methylation levels at the latest stages during seed maturation. Considering the delayed senescence phenotype of ddc, de-novo methylation via RdDM appears to be involved in initiation and execution of the senescence program. Furthermore, hypomethylation at ROS1 gene regulatory region was related to down regulation of gene expression. As an antagonist of RdDM, together with the early senescence phenotype of ros1, these results strengthen the importance of de-novo methylation for senescence, while active demethylation gets down regulated. Overall, methylation changes were little related to known gene expression changes that are associated with senescence. Limited targeting of WRKY and bZIP binding sites hinders conclusions about senescence specific effects of cytosine methylation in signal transduction networks. Altogether, the present work shines light on the importance of proper maintenance of cytosine methylation for flowering time, nutrient remobilization and senescence, and identifies defined cytosine methylation changes during senescence in a comprehensive physiological framework.
Site-dependent differences in DNA methylation and their impact on plant establishment in Populus trichocarpa
(2016) Schönberger, Brigitte; Ludewig, Uwe
Phosphate (Pi) limits total biomass production in natural tree ecosystems. Due to the low mobility of Pi in soil, higher plants, like trees, require special adaptations for phosphorus (P) acquisition. The genetic and physiological basis of this adaptation has been studied extensively. In addition, phosphorus starvation was recently suggested to affect epigenetic modifications in varying annual plant species. However, the impact of differential DNA methylation and microRNAs (miRNAs) on gene expression as well as site-dependent P-related physiology is largely unknown in perennials. In this study Populus trichocarpa clones, established from stem cuttings from two different locations, were grown in hydroponic culture with different P levels. Morphological and physiological parameters as well as, using bisulfite sequencing, site-specific genome-wide methylomes were determined. Gene and miRNA expression of differentially methylated regions was quantified via qPCR. Site-dependent differences in plant establishment were encountered, together with site-specific differentially methylated chromosomal regions. Methylation differences were nucleotide context-specific and extensively regulated miRNAs and their target genes in an organ-specific way. Though no direct relation between differential methylation in coding regions and their corresponding gene expression was observed, a general site-dependent transcriptional repression by DNA methylation was detected. Nevertheless, differential DNA methylation and gene expression was not affected by P nutrition, although recent studies described P-starvation induced DNA methylation changes, suggesting species-specific epigenetic mechanisms. However, differentially methylated miRNAs, together with their target genes, showed P-dependent expression profiles, indicating miRNA expression changes as a P-related epigenetic modification in poplar. Hence, it was shown that differences in DNA methylation or differentially methylated miRNAs might influence plant establishment and partially correlate with P acquisition, and thus be responsible for a site-dependent adaptation and growth performance, interesting for plant breeding, conservation biology and biodiversity studies of vegetatively propagated perennials.