Browsing by Subject "Methylierung"

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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.
Nutritional regulation of DNA methylation and gene expression in maize
(2018) Mager, Svenja; Ludewig, Uwe
DNA methylation in plants plays a role in transposon silencing, genome stability and gene expression regulation. Environmental factors alter the methylation pattern of DNA and recently nutrient stresses, such as phosphate starvation, were shown to alter DNA methylation. DNA methylation had been frequently addressed in plants with notably small genomes that are poor in transposons. Here, part of the DNA methylome of nitrogen-, phosphorus- and zinc-deficient (-N, -P and -Zn, respectively) maize roots were compared by reduced representation sequencing and their relationship with gene expression under prolonged stresses analyzed. Tremendous DNA methylation loss was encountered in maize under nitrogen and zinc deficiency, but much less under phosphorus deficiency. This occurred only in the symmetrical cytosine contexts, predominantly in CG context, but also in the CHG context. In contrast to other plants, differential methylation in the more flexible CHH context was essentially absent. For each sample, specific nutrient deficiency-regulated genes were differentially expressed. In -Zn samples the lowest number of differentially expressed genes was found while -N and -P samples contained a similar number of differentially expressed genes. For all samples, differentially methylated regions (DMRs) were predominantly identified in transposable elements (TEs). A minor fraction of such DMRs was associated with altered gene expression of nearby genes in -N and -P. Interestingly, although these TEs were mostly hypomethylated, they were associated with both upand down-regulated gene expression. For -Zn, these associations were not found but a correlation between hypomethylation of gene bodies and expression of some genes. Here again, hypomethylation occurred with up- and downregulation of gene expression. The results suggested a different methylome regulation in maize compared to rice and Arabidopsis upon nutrient deficiencies indicating a nutrient- and species-specific association of genomic DNA methylation and gene expression. The limited correlation between differential DNA methylation and gene expression suggested that heritable regulation of the expression of nutrient deficiency-regulated genes was not the primary function of the methylation loss. Rather, the major function of the DNA methylation loss in this experiment may have been to increase the genetic diversity in the next generation by increased frequency of recombination events, mutations and transposable element movements.
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.