Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors Franziska Meier-Stiegen1¤, Ralf Schwanbeck1*, Kristina Bernoth1, Simone Martini1, Thomas Hieronymus2, David Ruau 2 , Martin Zenke2, Ursula Just1* 1 Department of Biochemistry, Christian-Albrechts-University of Kiel, Kiel, Germany, 2 Department of Cell Biology, Institute for Biomedical Engineering, University Medical School, Rheinisch-Westfa¨lische Technische Hochschule Aachen, Aachen, Germany Abstract Background: Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. Methodology/Principal Findings: To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. Conclusion/Significance: We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context. Citation: Meier-Stiegen F, Schwanbeck R, Bernoth K, Martini S, Hieronymus T, et al. (2010) Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors. PLoS ONE 5(7): e11481. doi:10.1371/journal.pone.0011481 Editor: Jo-Ann L. Stanton, University of Otago, New Zealand Received March 18, 2010; Accepted June 10, 2010; Published July 8, 2010 Copyright: 2010 Meier-Stiegen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG SPP1109 to UJ (Ju197/5-1, 5-2), and TH and MZ (Ze432/2), SFB 415 project B8 to UJ and partially by DFG SPP1356 to MZ) and partially by Hensel Stiftung to UJ. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ujust@biochem.uni-kiel.de (UJ); rschwanbeck@biochem.uni-kiel.de (RS) ¤ Current address: Research Group Diabetes, University Hospital rechts der Isar, Munich Technical University, Munich, Germany Introduction The Notch signaling pathway is a highly conserved signaling mechanism that controls cell fate decisions, proliferation and apoptosis during development and in the adult [1,2]. In mammals, Notch proteins comprise a family of four transmembrane receptors (Notch1-4). Specific transmembrane ligands (Jagged-1, Jagged-2, Delta-like-1, Delta-like-3, and Delta-like-4) interact with Notch receptors on neighboring cells. Activating ligands induce cleavage near the transmembrane region of the Notch intracellular domain (NotchIC) resulting in the release and nuclear translocation of NotchIC [1]. Nuclear NotchIC interacts with the transcriptional repressor RBP-Jk (RBP-J/CSL/CBF1/Su(H)/Lag1), and converts it into an activator [3], leading to the expression of direct Notch target genes [4]. The outcome of Notch signaling is highly dependent on the cellular context [1]. Notch activity affects differentiation, prolifer- ation, and apoptotic programs in concert with other cell-intrinsic or cell-extrinsic developmental cues that are necessary to execute specific developmental programs [1]. However, despite the identification of many interacting pathways [4], it remains unclear how the highly variable, context-specific effects of Notch signaling are integrated at the molecular level, i.e. which specific target gene programs are activated. The best characterized direct targets of Notch signaling are the Hes (Hairy/Enhancer of Split) and Hey (also called Herp/Hesr/Hrt/ CHF/gridlock) families of basic helix-loop-helix (bHLH)-type transcriptional repressors [5,6]. Notch/RBP-J signaling activates Hes/Hey transcription, which leads to repression of Hes/Hey target genes such as tissue-specific transcriptional activators, thereby preventing differentiation [5]. More recently, several other genes with quite diverse functions have been found to be directly regulated by Notch signaling [7,8], implying that Notch exerts its pleiotropic functions by acting through multiple specific targets. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11481