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      Design Principles of Concentration-Dependent Transcriptome Deviations in Drug-Exposed Differentiating Stem Cells

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          Abstract

          Information on design principles governing transcriptome changes upon transition from safe to hazardous drug concentrations or from tolerated to cytotoxic drug levels are important for the application of toxicogenomics data in developmental toxicology. Here, we tested the effect of eight concentrations of valproic acid (VPA; 25–1000 μM) in an assay that recapitulates the development of human embryonic stem cells to neuroectoderm. Cells were exposed to the drug during the entire differentiation process, and the number of differentially regulated genes increased continuously over the concentration range from zero to about 3000. We identified overrepresented transcription factor binding sites (TFBS) as well as superordinate cell biological processes, and we developed a gene ontology (GO) activation profiler, as well as a two-dimensional teratogenicity index. Analysis of the transcriptome data set by the above biostatistical and systems biology approaches yielded the following insights: (i) tolerated (≤25 μM), deregulated/teratogenic (150–550 μM), and cytotoxic (≥800 μM) concentrations could be differentiated. (ii) Biological signatures related to the mode of action of VPA, such as protein acetylation, developmental changes, and cell migration, emerged from the teratogenic concentrations range. (iii) Cytotoxicity was not accompanied by signatures of newly emerging canonical cell death/stress indicators, but by catabolism and decreased expression of cell cycle associated genes. (iv) Most, but not all of the GO groups and TFBS seen at the highest concentrations were already overrepresented at 350–450 μM. (v) The teratogenicity index reflected this behavior, and thus differed strongly from cytotoxicity. Our findings suggest the use of the highest noncytotoxic drug concentration for gene array toxicogenomics studies, as higher concentrations possibly yield wrong information on the mode of action, and lower drug levels result in decreased gene expression changes and thus a reduced power of the study.

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          Most cited references 41

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          Master transcription factors and mediator establish super-enhancers at key cell identity genes.

          Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Super-enhancers in the control of cell identity and disease.

            Super-enhancers are large clusters of transcriptional enhancers that drive expression of genes that define cell identity. Improved understanding of the roles that super-enhancers play in biology would be afforded by knowing the constellation of factors that constitute these domains and by identifying super-enhancers across the spectrum of human cell types. We describe here the population of transcription factors, cofactors, chromatin regulators, and transcription apparatus occupying super-enhancers in embryonic stem cells and evidence that super-enhancers are highly transcribed. We produce a catalog of super-enhancers in a broad range of human cell types and find that super-enhancers associate with genes that control and define the biology of these cells. Interestingly, disease-associated variation is especially enriched in the super-enhancers of disease-relevant cell types. Furthermore, we find that cancer cells generate super-enhancers at oncogenes and other genes important in tumor pathogenesis. Thus, super-enhancers play key roles in human cell identity in health and in disease. Copyright © 2013 Elsevier Inc. All rights reserved.
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              Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

              Current neural induction protocols in human ES cells (hESCs) rely on embryoid body formation, stromal feeder co-culture, or selective survival conditions; each strategy displaying significant drawbacks such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient for inducing rapid and complete neural conversion of hESCs under adherent culture conditions. Temporal fate analysis reveals a transient FGF5+ epiblast-like stage followed by PAX6+ neural cells competent of rosette formation. Initial cell density determines the ratio of CNS versus neural crest progeny. Directed differentiation of human iPSCs into midbrain dopamine and spinal motoneurons confirm robustness and general applicability of the novel induction protocol. Noggin/SB431542 based neural induction should greatly facilitate the use of hESC and hiPSCs in regenerative medicine and disease modeling and obviate the need for stromal feeder or embryoid body based protocols.
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                Author and article information

                Affiliations
                []Doerenkamp-Zbinden Chair for in Vitro Toxicology and Biomedicine, University of Konstanz , 78457 Konstanz, Germany
                []Department of Statistics, TU Dortmund , D-44221 Dortmund, Germany
                [§ ]OÜ Quretec (Qure), Limited Liability Company, 51003 Tartu, Estonia
                []Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne (UKK) , D-50931 Cologne, Germany
                []Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund , D-44139 Dortmund, Germany
                Author notes
                [* ]University of Konstanz, Box 657, D-78457 Konstanz, Germany. Tel: +49-7531-884181. E-mail: Tanja.Waldmann@ 123456uni-konstanz.de .
                Journal
                Chem Res Toxicol
                Chem. Res. Toxicol
                tx
                crtoec
                Chemical Research in Toxicology
                American Chemical Society
                0893-228X
                1520-5010
                02 January 2014
                17 March 2014
                : 27
                : 3 , Systems Toxicology
                : 408-420
                24383497 3958134 10.1021/tx400402j
                Copyright © 2014 American Chemical Society
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                Article
                Custom metadata
                tx400402j
                tx-2013-00402j

                Toxicology

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