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Debojyoti Chakraborty,
Dennis Kappei,
Mirko Theis, Anja Nitzsche,
Li Ding,
Maciej Paszkowski-Rogacz,
Vineeth Surendranath,
Nicolas Berger,
Herbert Schulz,
Kathrin Saar,
Norbert Hubner,
Frank Buchholz
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ABSTRACT: Whereas methods to comprehensively study cellular roles of protein-coding genes are available, techniques to systematically investigate long noncoding RNAs (lncRNAs), which have been implicated in diverse biological pathways, are limited. Here we report combined knockdown and localization analysis of noncoding RNAs (c-KLAN) that merges functional characterization and localization approaches to study lncRNAs. Using this technique we identified transcripts that regulate mouse embryonic stem cell identity.
Nature Methods 02/2012; 9(4):360-2. · 19.28 Impact Factor
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ABSTRACT: The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)--originally developed for Alzheimer's disease--are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)--a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.
PLoS ONE 01/2011; 6(4):e18709. · 4.09 Impact Factor
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Anja Nitzsche,
Maciej Paszkowski-Rogacz,
Filomena Matarese,
Eva M Janssen-Megens,
Nina C Hubner,
Herbert Schulz,
Ingrid de Vries,
Li Ding,
Norbert Huebner,
Matthias Mann,
Hendrik G Stunnenberg,
Frank Buchholz
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ABSTRACT: For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21--pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.
PLoS ONE 01/2011; 6(5):e19470. · 4.09 Impact Factor
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Li Ding,
Maciej Paszkowski-Rogacz, Anja Nitzsche,
Mikolaj Michal Slabicki,
Anne-Kristin Heninger,
Ingrid de Vries,
Ralf Kittler,
Magno Junqueira,
Andrej Shevchenko,
Herbert Schulz, [......],
Agapios Sachinidis,
Juergen Hescheler,
Roberto Iacone,
Konstantinos Anastassiadis,
A Francis Stewart,
M Teresa Pisabarro,
Antonio Caldarelli,
Ina Poser,
Mirko Theis,
Frank Buchholz
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ABSTRACT: Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.
Cell stem cell 05/2009; 4(5):403-15. · 23.56 Impact Factor
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Ina Poser,
Mihail Sarov,
James R A Hutchins,
Jean-Karim Hériché,
Yusuke Toyoda,
Andrei Pozniakovsky,
Daniela Weigl, Anja Nitzsche,
Björn Hegemann,
Alexander W Bird, [......],
Youming Zhang,
Kim Nasmyth,
Kevin P White,
Steffen Dietzel,
Karl Mechtler,
Richard Durbin,
A Francis Stewart,
Jan-Michael Peters,
Frank Buchholz,
Anthony A Hyman
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ABSTRACT: The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.
Nature Methods 06/2008; 5(5):409-15. · 19.28 Impact Factor
