TY  - JOUR
A1  - Huang, Bei
A1  - Belharazem, Djeda
A1  - Li, Li
A1  - Kneitz, Susanne
A1  - Schnabel, Philipp A.
A1  - Rieker, Ralf J.
A1  - Körner, Daniel
A1  - Nix, Wilfried
A1  - Schalke, Berthold
A1  - Müller-Hermelink, Hans Konrad
A1  - Ott, German
A1  - Rosenwald, Andreas
A1  - Ströbel, Philipp
A1  - Marx, Alexander
T1  - Anti-apoptotic signature in thymic squamous cell carcinomas – functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c
JF  - Frontiers in Oncology
N2  - The molecular pathogenesis of thymomas and thymic arcinomas (TCs) is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and TCs, suggesting that other oncogenic principles might be important.This made us re-analyze historic expression data obtained in a spectrumof thymomas and thymic squamous cell carcinomas (TSCCs) with a custom-made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC.
KW  - gene expression
KW  - MTCH2
KW  - targeted
KW  - myasthenia gravis
KW  - apoptosis
KW  - thymus
KW  - thymoma
KW  - thymic carcinoma
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132214
VL  - 3
IS  - 316
ER  - 
TY  - JOUR
A1  - Schlereth, Katharina
A1  - Heyl, Charlotte
A1  - Krampitz, Anna-Maria
A1  - Mernberger, Marco
A1  - Finkernagel, Florian
A1  - Scharfe, Maren
A1  - Jarek, Michael
A1  - Leich, Ellen
A1  - Rosenwald, Andreas
A1  - Stiewe, Thorsten
T1  - Characterization of the p53 Cistrome - DNA Binding Cooperativity Dissects p53's Tumor Suppressor Functions
JF  - PLOS Genetics
N2  - p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context-and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53's apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing.
KW  - cell-cycle arrest
KW  - gene expression
KW  - breast cancer
KW  - human genome
KW  - transcriptional repression
KW  - consensus DNA
KW  - in-vivo
KW  - apoptosis
KW  - network
KW  - damage
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127579
SN  - 1553-7404
VL  - 9
IS  - 8
ER  - 
TY  - JOUR
A1  - Mueller, Kerstin
A1  - Quandt, Jasmin
A1  - Marienfeld, Ralf B.
A1  - Weihrich, Petra
A1  - Fiedler, Katja
A1  - Claussnitzer, Melina
A1  - Laumen, Helmut
A1  - Vaeth, Martin
A1  - Berberich-Siebelt, Frederike
A1  - Serfling, Edgar
A1  - Wirth, Thomas
A1  - Brunner, Cornelia
T1  - Octamer-dependent transcription in T cells is mediated by NFAT and \(NF-\kappa B\)
JF  - Nucleic Acids Research
N2  - The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and \(NF-\kappa B\)-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/\(NF-\kappa B\) sites. An array of genetic and biochemical analyses illustrates the involvement of the \(Ca^{2+}\)/calmodulin-dependent phosphatase calcineurin as well as NFAT and \(NF-\kappa B\) transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or \(NF-\kappa B\) transcription factors.
KW  - germinal center formation
KW  - OBF-1 OCA-B
KW  - coactivator OBF-1
KW  - gene expression
KW  - functional characterization
KW  - immunoglobulin promoters
KW  - OCT-1-deficient mice
KW  - embryonic lethality
KW  - endothelial cells
KW  - murine homolog
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123280
SN  - 1362-4962
VL  - 41
IS  - 4
ER  - 
TY  - THES
A1  - Hock, Matthias
T1  - Analyse der NFATc1-Genexpression durch eGFP-BAC-Reportermäuse
T1  - Analysis of NFATc1 gene expression using eGFP-BAC reporter mice
N2  - In Lymphozyten wird nach Antigenaktivierung die Expression des Nfatc1-Gens durch Aktivierung des P1-Promoters stark induziert. Dagegen ist die, durch den Promoter P2 vermittelte Expression ebenso wie die der anderen NFAT Faktoren c2 und c3 konstitutiv. Die Akkumulation der dabei gebildeten Isoform NFATc1/αA ist sowohl für Effektorfunktionen wie die Zytokinproduktion sowie die Proliferation und das Überleben der aktivierten Zellen wichtig (Chuvpilo et al., 2002). Um die Expression des Nfatc1-Gens auf Einzelzellebene messen zu können, wurden BAC (bacterial artificial chromosom) transgene Mauslinien generiert, die einen 210kb großen Bereich des Nfatc1-Gens der Maus enthalten. In diesen Lokus wurde ein eGFP-Reportergen innerhalb des allen Isoformen gemeinsamen, dritten Exons integriert. In dieser Arbeit wird durch semiquantitative RT-PCR-Experimente von Gesamt-Milzzellen und TLymphozyten gezeigt, dass in den B6/NFATc1-eGFP-BAC-Reportermäusen die Expression der eGFP-cDNA analog zum endogenen Nfatc1-Lokus der Kontrolle der beiden Promotoren P1 und P2 unterliegt. In Western Blot Experimenten wird in diesen Zellen mittels eines NFATc1α-spezifischen Antikörpers eine induzierbare und CsA-sensitive α-GFP-Isoform - vergleichbar mit der endogenen NFATc1α-Isoform - nachgewiesen. Gleichzeitig zeigen NFATc1-Antikörper das konstitutiv exprimierte GFPβ-Protein. Die Korrelation der Expression von NFATc1 und GFP auf mRNA- und Proteinebene machen in B6/NFATc1-eGFP-BAC-Reportermäusen das GFP-Protein somit zu einem sensitiven und spezifischen Marker der NFATc1-Aktivität. In FACS-Analysen gibt der Anstieg der GFP-Fluoreszenzintensität bei Stimulation von Gesamt- Milzzellen bzw. T-Lymphozyten um bis auf das Dreifache die Induktion von NFATc1 wider. Unter dem Einfluss von CsA verbleibt die GFPFluoreszenzintensität auf dem Niveau unstimulierter Zellen. Die GFPFluoreszenz korreliert darüber hinaus bei Primärstimulation mit der Expression des IL-2-Gens, dessen Promotor mit 5 NFAT-Bindestellen den Prototyp eines NFATc1-Targets darstellt (Serfling et al., 1989). Die Analyse der Koexpression von NFATc1 und GFP mittels Fluoreszenzmikroskopie zeigt in allen stimulierten, GFP-positiven CD4+-Lymphozyten die nukleäre Lokalisation von 75 NFATc1, vor allem von NFATc1α. Die Analyse des GFP-Phänotyps in alloreaktiven T-Zellen zeigt bei Abstoßungsreaktionen in vitro („Mixed Lymphocyte Reactions“) eine selektive Zunahme der Fluoreszenz dieser Zellen um bis auf das Vierfache, was die Rolle von NFATc1 für die Effektorfunktion aktivierter T-Lymphozyten verdeutlicht. GFP und das endogene NFATc1 werden bei Stimulation konventioneller T-Zellen (Tcons, CD4+CD25-FoxP3-) stark exprimiert, während natürliche regulatorische T-Zellen (nTregs, CD4+CD25+FoxP3+) konstant geringe NFATc1- und GFP-Konzentrationen zeigen. In induzierten regulatorischen T-Lymphozyten (iTregs) supprimiert TGF- β konzentrationsabhängig die GFP-Fluoreszenz bis auf das Niveau unstimulierter Lymphozyten. Während in nTregs die Suppression des Nfatc1- Gens im wesentlichen durch FoxP3 erfolgt (Torgerson et al., 2009), scheint dies in iTregs vor allem über den TGF-β Signalweg vermittelt zu werden. Die Analyse der GFP-Expression in den verschiedenen Stadien der TZellentwicklung zeigt weiterhin deutliche Unterschiede in der Aktivität des Nfatc1-Gens. Dies wird durch die starke Aktivität des BAC-Genlokus in CD4- CD8- DN Thymozyten, welche eine sechsfach höhere GFP-Expression aufweisen als CD4+CD8+ DP Zellen, deutlich.
N2  - Activation of lymphocytes causes a high level of Nfatc1 due to P1-promoter induction, whereas the P2-promoter leads to an constitutive expression of NFATc2/c3. NFATc1/αA is essential for effector-function, proliferation and survival of activated cells (Chuvpilo et al., 2002). Here we show that the eGFP-cDNA integrated in a NFATc1-eGFP-BAC-construct is under control of both promoters (P1, P2) and correlates with the NFATc1-Expression on mRNA and protein-levels. In FACS-analysis there is an increase in eGFP-fluorescence intensity, which is highly CsA-sensitive. In natural and induced Tregs we observed a low level of eGFP-expression correlating with concentration of TGF-β. CD4-CD8- DN cells show the highest eGFP-Expression in thymocytes.
KW  - NFATc1
KW  - Genexpression
KW  - eGFP
KW  - BAC-Konstrukt
KW  - NFATc1
KW  - Genexpression
KW  - eGFP
KW  - BAC-Konstrukt
KW  - NFATc1
KW  - gene expression
KW  - eGFP
KW  - BAC-construct
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-80596
ER  -