Center for Biomedical Genetics


Center for Biomedical Genetics

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Elkon R.,Netherlands Cancer Institute | Zlotorynski E.,Netherlands Cancer Institute | Agami R.,Netherlands Cancer Institute | Agami R.,Center for Biomedical Genetics
BMC Genomics | Year: 2010

Background: mRNA levels in cells are determined by the relative rates of RNA production and degradation. Yet, to date, most analyses of gene expression profiles were focused on mechanisms which regulate transcription, while the role of mRNA stability in modulating transcriptional networks was to a large extent overlooked. In particular, kinetic waves in transcriptional responses are usually interpreted as resulting from sequential activation of transcription factors.Results: In this study, we examined on a global scale the role of mRNA stability in shaping the kinetics of gene response. Analyzing numerous expression datasets we revealed a striking global anti-correlation between rapidity of induction and mRNA stability, fitting the prediction of a kinetic mathematical model. In contrast, the relationship between kinetics and stability was less significant when gene suppression was analyzed. Frequently, mRNAs that are stable under standard conditions were very rapidly down-regulated following stimulation. Such effect cannot be explained even by a complete shut-off of transcription, and therefore indicates intense modulation of RNA stability.Conclusion: Taken together, our results demonstrate the key role of mRNA stability in determining induction kinetics in mammalian transcriptional networks. © 2010 Elkon et al; licensee BioMed Central Ltd.

Li V.S.W.,University Utrecht | Ng S.S.,University Utrecht | Boersema P.J.,University Utrecht | Boersema P.J.,Netherlands Proteomics Center | And 15 more authors.
Cell | Year: 2012

Degradation of cytosolic β-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that β-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound β-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses β-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-β-catenin. Subsequently, newly synthesized β-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors. © 2012 Elsevier Inc. © 2012 Elsevier Inc.

Agami R.,Netherlands Cancer Institute | Agami R.,Center for Biomedical Genetics
European Journal of Clinical Investigation | Year: 2010

microRNAs (miRNAs) are genes involved in normal development and cancer. They inhibit gene expression through interaction with 3′-untranslated regions of messenger RNAs, and are thought to control the expression of a large proportion of the protein-coding genome. However, it is becoming apparent that RNA-binding proteins (RBPs) control the biogenesis of miRNAs, their activity and stability. This indicates the existence of interplay between RBPs and miRNAs that affects gene expression and processes ranging from development, maintenance of stem cell phenotype and stress responses. Although miRNAs are prominent factors in cancer, little is known about how RBPs affect their cancerous function. © 2010 Stichting European Society for Clinical Investigation Journal Foundation.

Roukens M.G.,Leiden University | Alloul-Ramdhani M.,Leiden University | Baan B.,Leiden University | Kobayashi K.,Leiden University | And 6 more authors.
Nature Cell Biology | Year: 2010

We show that the transcriptional repressor Tel plays an evolutionarily conserved role in angiogenesis: it is indispensable for the sprouting of human endothelial cells and for normal development of the Danio rerio blood circulatory system. Tel orchestrates endothelial sprouting by binding to the generic co-repressor, CtBP. The Tel-CtBP complex temporally restricts a VEGF (vascular endothelial growth factor)-mediated pulse of dll4 expression and thereby directly links VEGF receptor intracellular signalling and intercellular Notch-Dll4 signalling. It further controls branching by regulating expression of other factors that constrain angiogenesis such as sprouty family members and ve-cadherin. Thus, the Tel-CtBP complex conditions endothelial cells for angiogenesis by controlling the balance between stimulatory and antagonistic sprouting cues. Tel control of branching seems to be a refinement of invertebrate tracheae morphogenesis that requires Yan, the invertebrate orthologue of Tel. This work highlights Tel and its associated networks as potential targets for the development of therapeutic strategies to inhibit pathological angiogenesis. © 2010 Macmillan Publishers Limited. All rights reserved.

Ghamari A.,Erasmus Medical Center | van de Corput M.P.C.,Erasmus Medical Center | Thongjuea S.,University of Bergen | Van Cappellen W.A.,Erasmus Medical Center | And 8 more authors.
Genes and Development | Year: 2013

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments. © 2013 by Cold Spring Harbor Laboratory Press.

Van Haaften G.,Netherlands Cancer Institute | Agami R.,Center for Biomedical Genetics
Genes and Development | Year: 2010

The miR-17-92 gene cluster, with its six different mature microRNAs (miRNAs), has an established oncogenic function. However, the oncogenic contribution of each individual miRNA in the cluster has not been assigned. Two studies published in the December 15, 2009, issue of Genes & Development by Mu and colleagues (pp. 2806-2811) and Olive and colleagues (pp. 2839-2849) dissected the miR-17-92 cluster to its individual miRNA components and identified their relative contributions to oncogenic transformation in mouse model systems. © 2010 by Cold Spring Harbor Laboratory Press.

Melo C.A.,Netherlands Cancer Institute | Melo C.A.,Center for Neuroscience and Cell Biology | Drost J.,Netherlands Cancer Institute | Wijchers P.J.,University Utrecht | And 11 more authors.
Molecular Cell | Year: 2013

Binding within or nearby target genes involved in cell proliferation and survival enables the p53 tumor suppressor gene to regulate their transcription and cell-cycle progression. Using genome-wide chromatin-binding profiles, we describe binding of p53 also to regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intrachromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation. Furthermore, p53BERs produce, in a p53-dependent manner, enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arrest. Thus, our results ascribe transcription enhancement activity to p53 with the capacity to regulate multiple genes from a single genomic binding site. Moreover, eRNA production from p53BERs is required for efficient p53 transcription enhancement. © 2013 Elsevier Inc.

Jenal M.,Netherlands Cancer Institute | Elkon R.,Netherlands Cancer Institute | Loayza-Puch F.,Netherlands Cancer Institute | Van Haaften G.,Netherlands Cancer Institute | And 9 more authors.
Cell | Year: 2012

Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3′ untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA. © 2012 Elsevier Inc.

Petersen M.,Center for Biomedical Genetics | Petersen M.,Leiden University | Pardali E.,Center for Biomedical Genetics | Van Der Horst G.,Leiden University | And 4 more authors.
Oncogene | Year: 2010

Transforming growth factor (TGF)-Β can suppress and promote breast cancer progression. How TGF-Β elicits these dichotomous functions and which roles the principle intracellular effector proteins Smad2 and Smad3 have therein, is unclear. Here, we investigated the specific functions of Smad2 and Smad3 in TGF-Β-induced responses in breast cancer cells in vitro and in a mouse model for breast cancer metastasis. We stably knocked down Smad2 or Smad3 expression in MDA-MB-231 breast cancer cells. The TGF-Β-induced Smad3-mediated transcriptional response was mitigated and enhanced by Smad3 and Smad2 knockdown, respectively. This response was also seen for TGF-Β-induced vascular endothelial growth factor (VEGF) expression. TGF-Β induction of key target genes involved in bone metastasis, were found to be dependent on Smad3 but not Smad2. Strikingly, whereas knockdown of Smad3 in MDA-MB-231 resulted in prolonged latency and delayed growth of bone metastasis, Smad2 knockdown resulted in a more aggressive phenotype compared with control MDA-MB-231 cells. Consistent with differential effects of Smad knockdown on TGF-Β-induced VEGF expression, these opposing effects of Smad2 versus Smad3 could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo. Thus, Smad2 and Smad3 differentially affect breast cancer bone metastasis formation in vivo. © 2010 Macmillan Publishers Limited All rights reserved.

Van de Corput M.P.C.,Center for Biomedical Genetics | De Boer E.,Center for Biomedical Genetics | Knoch T.A.,Center for Biomedical Genetics | Van Cappellen W.A.,Erasmus Medical Center | And 3 more authors.
Journal of Cell Science | Year: 2012

The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the β-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the β-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and superresolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping. © 2012.

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