Komplementationsstudien der cpd-Mutante und Analyse der CPD-Interaktionspartner mittels reverser Genetik

Anhand von Komplementationsstudien der cpd-Mutante konnte die Funktionalitaet eines CPD-GFP-Fusionsproteins in planta demonstriert werden. Die Komplementation erfolgte unter Kontrolle des endogenen CPD-Promotors und unter Kontrolle des konstitutiven CaMV-35S-Promotors. Die transgenen Pflanzen wurden zur zellulaeren und gewebespezifischen Lokalisation des CPD-Proteins verwendet. Die Lokalisation des CPD-Proteins an der ER-Membran konnte sowohl ueber die Detektion der GFP-Fluoreszenz des generierten CPD-Fusionsproteins als auch mit einer Westen-Blot-Analyse eines differentiell zentrifugierten Zellextrakts bestaetigt werden. Konditionale Komplementationsstudien der cpd-Mutante verdeutlichen die wichtige Rolle von Brassinosteroiden bei der Pflanzenentwicklung. Durch konditionale, biochemische Komplementationen wurde demonstriert, daß Brassinosteroide kontinuierlich waehrend der Pflanzenentwicklung benoetigt werden. Durch die konditionale genetische Komplementation der cpd-Mutante mit einem Oestradiol-induzierbaren Komplementationskonstrukt und der Verwendung eines unabhaengigen, induzierbaren GUS-Reporterkonstruktes war es moeglich einen Steroidtransport in planta nachzuweisen. Anhand des induziebaren GUS-Konstruktes wurde die inhomogene Oestradiol-Verteilung nach der lokal begrenzten Applikation mit Oestradiol-haltiger Lanolinpaste gezeigt. Verschiedene Studien belegen den Transport des Oestradiols. Die entsprechende lokale Komplementation der cpd-Mutante hat eine ganzheitliche Reaktion der Pflanze gezeigt, was einen Transport des Syntheseendprodukts Brassinolid oder eines nachgeschalteten Signals belegt. Die Analyse der CPD-Interaktionspartner ueber reverse Genetik hat zur Identifikation der iap2-1-Mutante gefuehrt, die eine T-DNA-Insertion in einem, fuer ein Ringfingerprotein-codierendes Gen traegt. Die Mutante weist einen vergleichbaren Phaenotyp wie CPD-Ueberexpressionslinien auf, die auf Kontrollmedium ein verstaerktes Wurzelwachstum entwickeln. Die iap2-1-Mutante zeigt auf Hochsteroidmedium eine hypersensitive Reaktion gegenueber Brassinolid, was auf einen veraenderten, endogenen Steroidgehalt oder eine modulierte Signaltransduktion hindeutet. Diese Ergebnisse stehen im Einklang mit der postulierten Funktion des IAP2-Ringfingerproteins, das als E3-Ubiquitin-Ligase an der Regulation der CPD-Proteinstabilitaet beteiligt sein kann. Es bleibt aber offen, ob IAP2 an der direkten Regulation des CPD-Proteins oder einer anderen Komponente der Brassinosteroidsignaltransduktion beteiligt ist. Die Analyse von T-DNA-Insertionsmutanten in fuenf OBP-Genen hat nicht zur Identifikation eines Steroidphaenotyps gefuehrt. Damit zeichnet sich eine redundante Funktionalitaet innerhalb der OBP-Proteinfamilie aus A. thaliana ab, wie sie z. B. bei den homologen OSH-Proteinen in Hefe beobachtet wurde ab. Analysen von Doppel- und Mehrfachmutanten der CPD-Interaktionspartner und insbesondere der obp-Mutanten werden ein genaueres Bild der IAP und OBP-Funktion geben

Dynamic Regulation of H3K27 Trimethylation during Arabidopsis Differentiation

During growth of multicellular organisms, identities of stem cells and differentiated cells need to be maintained. Cell fate is epigenetically controlled by the conserved Polycomb-group (Pc-G) proteins that repress their target genes by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although H3K27me3 is associated with mitotically stable gene repression, a large fraction of H3K27me3 target genes are tissue-specifically activated during differentiation processes. However, in plants it is currently unclear whether H3K27me3 is already present in undifferentiated cells and dynamically regulated to permit tissue-specific gene repression or activation. We used whole-genome tiling arrays to identify the H3K27me3 target genes in undifferentiated cells of the shoot apical meristem and in differentiated leaf cells. Hundreds of genes gain or lose H3K27me3 upon differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3 is correlated with gene repression, and its release preferentially results in tissue-specific gene activation, both during differentiation and in Pc-G mutants. We further reveal meristem- and leaf-specific targeting of individual gene families including known but also likely novel regulators of differentiation and stem cell regulation. Interestingly, H3K27me3 directly represses only specific transcription factor families, but indirectly activates others through H3K27me3-mediated silencing of microRNA genes. Furthermore, H3K27me3 targeting of genes involved in biosynthesis, transport, perception, and signal transduction of the phytohormone auxin demonstrates control of an entire signaling pathway. Based on these and previous analyses, we propose that H3K27me3 is one of the major determinants of tissue-specific expression patterns in plants, which restricts expression of its direct targets and promotes gene expression indirectly by repressing miRNA genes

Tissue effects of a newly developed diode pumped pulsed Thulium:YAG laser compared to continuous wave Thulium:YAG and pulsed Holmium:YAG laser

Purpose!#!The objective of this study is to evaluate the laser-tissue effects of laser radiation emitted by a newly developed high frequency pulsed Tm:YAG laser in comparison to the continuous wave Tm:YAG laser and the pulsed Ho:YAG laser.!##!Methods!#!Ex-vivo experiments were performed on freshly slaughtered porcine kidneys in a physiological saline solution. Experiments were performed using two different laser devices in different settings: A Tm:YAG laser was operated in a pulsed mode up to 300 Hz and in a continuous wave (CW) mode. Results were compared with a 100 W standard pulsed Ho:YAG laser system. Comparative tissue experiments were performed at 5 W, 40 W and 80 W. The incision depth and the laser damage zone were measured under a microscope using a calibrated ocular scale.!##!Results!#!Increased laser power resulted in increased incision depth and increased laser damage zone for all investigated lasers in this set-up. The Ho:YAG created the largest combined tissue effect at the 5 W power setting and seems to be the least controllable laser at low power for soft tissue incisions. The CW Tm:YAG did not incise at all at 5 W, but created the largest laser damage zone. For the new pulsed Tm:YAG laser the tissue effect grew evenly with increasing power.!##!Conclusion!#!Among the investigated laser systems in this setting the pulsed Tm:YAG laser shows the most controllable behavior, insofar as both the incision depth and the laser damage zone increase evenly with increasing laser power

DNA methylation of tumor associated calcium signal transducer 2 (TACSTD2) loci shows association with clinically aggressive renal cell cancers

Background!#!DNA methylation is frequently observed in the development and progression of many human tumors as well as renal cell cancer (RCC). Tumor Associated Calcium Signal Transducer 2 (TACSTD2) participates in cell cycle progression through MAPK signalling pathway activation. Moreover, tumor-specific hypermethylation and association with aggressive cancer characteristics has been found for lung adenocarcinoma, hepatocellular carcinoma and cholangiocarcinoma. Whether TACSTD2 is tumor specifically hypermethylated in RCC or shows association of methylation with adverse clinicopathological parameters and survival of patients has not been investigated at yet.!##!Methods!#!Quantitative methylation-specific PCR (qMSP) analysis of a locus in the intron 1 region of TACSTD2 gene was carried out in a cross-sectional study of 127 paired RCC and normal samples. In silico analysis of TACSTD2 methylation in the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) dataset of 280 patients served as validation cohort. Statistical analyses were carried out using the two-sided paired t-test for matched tumor and normal sample comparisons, logistic regression for subgroup comparisons, Cox regression for analysis of recurrence free survival (RFS) and Pearson correlation analysis for correlation of TACSTD2 methylation and TACSTD2 mRNA in KIRC data.!##!Results!#!Higher methylation levels in RCC were significantly associated with advanced disease (p < 0.001), high tumor stage (p = 0.003), tumor differentiation (p = 0.033) and presence of lymph node (p = 0.021) or distant metastases (p = 0.008). TACSTD2 hypermethylation was associated with a shorter RFS of patients and demonstrate statistical independency from clinical parameters as state of metastasis, tumor stage, grade and state of advanced disease. In silico validation using TCGA KIRC data also demonstrated association of TACSTD2 loci with adverse clinicopathology and shortened RFS of patients. In addition, in silico analyses of TCGA KIRC data showed an inverse correlation between DNA methylation levels of TACSTD2 and mRNA expression.!##!Conclusions!#!Our results suggest an association between TACSTD2 methylation and disease progression and clinical course of RCC

Identification of a Novel Renal Metastasis Associated CpG-Based DNA Methylation Signature (RMAMS)

Approximately 21% of patients with renal cell cancer (RCC) present with synchronous metastatic disease at the time of diagnosis, and metachronous metastatic disease occurs in 20–50% of cases within 5 years. Recent advances in adjuvant treatment of aggressive RCC following surgery suggest that biomarker-based prediction of risk for distant metastasis could improve patient selection. Biometrical analysis of TCGA-KIRC data identified candidate loci in the NK6 homeobox 2 gene (NKX6-2) that are hypermethylated in primary metastatic RCC. Analyses of NKX6-2 DNA methylation in three gene regions including a total of 16 CpG sites in 154 tumor-adjacent normal tissue, 189 RCC, and 194 metastatic tissue samples from 95 metastasized RCC patients revealed highly significant tumor-specific, primary metastatic-specific, and metastatic tissue-specific hypermethylation of NKX6-2. Combined CpG site methylation data for NKX6-2 and metastasis-associated genes (INA, NHLH2, and THBS4) demonstrated similarity between metastatic tissues and metastatic primary RCC tissues. The random forest method and evaluation of an unknown test cohort of tissues using receiver operator characteristic curve analysis revealed that metastatic tissues can be differentiated by a median area under the curve of 0.86 (p = 1.7 × 10−8–7.5 × 10−3) in 1000 random runs. Analysis of variable importance demonstrated an above median contribution for decision-making of at least one CpG site in each of the genes, suggesting superior informativity for sites annotated to NHLH2 and NKX6-2. Thus, DNA methylation of NKX6-2 is associated with the metastatic state of RCC tissues and contributes to a four-gene-based statistical predictor of tumoral and metastatic renal tissues

DNA Methylation in <i>INA</i>, <i>NHLH2</i>, and <i>THBS4</i> Is Associated with Metastatic Disease in Renal Cell Carcinoma

The detection of DNA methylation in primary tumor tissues could be relevant for early stratification of aggressive renal cell carcinomas (RCCs) as a basis for future personalized adjuvant therapy. Methylated TCGA KIRC based candidate CpG loci in INA, NHLH2, and THBS4 that are possibly associated with RCC metastasis were evaluated by pyrosequencing in 154 paired normal adjacent and primary tumor tissues, as well as in 202 metastatic tissues. Statistical analysis was carried out by bivariate logistic regression for group comparisons, log rank survival analysis, and unsupervised and supervised analysis for the classification of tumors. Increased methylation of INA, NHLH2, and THBS4 loci were significantly associated with distant metastasis in primary tumors (p p = 7.88 × 10−8, 5.57 × 10−10, 2.06 × 10−7) and tumor tissues (p = 3.72 × 10−24, 3.17 × 10−13, 1.58 × 10−19), and shortened progression free survival in patients (p = 0.03). Combined use of CpG site-specific methylation permits the discrimination of tissues with metastatic disease and reveals a significant contribution of CpG sites in all genes to the statistical classification model. Thus, metastasis in RCC is significantly associated with methylation alterations in INA, NHLH2, and THBS4 loci, providing independent information for the potential early detection of aggressive renal cancers as a rationale for stratifying patients to adjuvant therapies

C-23 Hydroxylation by Arabidopsis CYP90C1 and CYP90D1 Reveals a Novel Shortcut in Brassinosteroid Biosynthesis

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)–catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5α-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5α-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol

Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance

Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated tEpe1 which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non-cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell-integrity MAP kinase stress signalling pathway, mutations in which alter resistance. Thus, environmental changes elicit a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered