Context-Specific Notch Targets Figure 4. Induction heat maps of Notch1 target genes involved in transcription, apoptosis, cell cycle and cell differentiation. The identified Notch1 targets were grouped into different Gene Ontology (GO) terms using the DAVID functional annotation analysis tool (http://david. abcc.ncifcrf.gov/). Note that only differentially regulated genes by Notch1 are shown as Log2 transformed values. If no gene symbol was available for the regulated transcript, GenBank accession number was given in parentheses. Significant changes of differentially regulated gene expression according to the criteria outlined in Material and Methods were labeled with a black or white dot for up- and down-regulated genes, respectively. Redundant probe sets for the same gene were removed except when significantly different results were obtained (e.g. Sox9). Pcdha@*) indicates the protocadherin acluster comprising Pcdha1, Pcdha2, Pcdha3, Pcdha4, Pcdha5, Pcdha6, Pcdha7, Pcdha8, Pcdha9, Pcdha10, Pcdha11, Pcdha12, Pcdhac1, Pcdhac2, and Pcdhgb2. The complete list of regulated genes is available in the Supplement Table S1. doi:10.1371/journal.pone.0011481.g004 differentiation (Fig. 3). Only about 10% of all identified Notch1 regulated genes were common for the different cell populations and culture conditions analyzed (Fig. 3). These results demonstrate that Notch/RBP-J signaling regulates different sets of genes in different cell types. Furthermore, other signaling pathways induced by the culture supplements strongly influence which target genes are activated by Notch/RBP-J signaling. To validate further our target gene identification, we compared the Notch1 target genes identified in ESC cultured in media favoring ectodermal differentiation (ESCe) with the Notch1 target genes identified recently in another study in ESC cultured under similar culture conditions [19]. About 44% of the activated genes in our ESCe analysis were also activated in the study by Main et al [19], thus revealing a considerable overlap of Notch1 targets in the same cell type and under similar differentiation conditions despite the use of different ESC (EB5 vs. 46C), different Notch1 induction systems (induction of nuclear translocation of the NotchIC-ERT fusion protein by OHT vs. induction of NotchIC expression by the Tet system) and different time point of analyses (4 h induction vs. 6 h induction). The high overlap of Notch1 target genes identified in similar cell type and culture conditions in two separate studies with different approaches of Notch activation together with the high cell type and context dependency of Notch1 target genes identified in the present study suggests that Notch target gene induction is tightly regulated, despite its cell-context dependency. Genes targeted by activated Notch1 encompassed a wide variety of intracellular and extracellular regulatory proteins, including cell lineage determinants, transcription factors, cell cycle regulators, intracellular signaling mediators and receptors and ligands, reflecting the pleiotropic functions of Notch (Fig. 4, 5, 6). In line with the short time of induction, most genes were regulated by activated Notch1 in the presence of CHX as well and thus present likely direct target genes of Notch1 (Fig. 4, 5, 6). To confirm our gene expression array data, 30 genes that were differentially regulated by Notch1 signaling in our array analyses were examined by qPCR. Gene expression values of EB5 NERT and EB5 control cells under differentiating (ESCe and ESCm) and self- renewing conditions (ESCs) and in mesodermal cells (Mesoderm) grown in the presence or absence of OHT and in the presence or absence of CHX for 4 h were obtained (Fig. 7). In line with our array data, none of the genes analyzed was regulated by OHT in EB5 control cells and all showed a comparable pattern of regulation in EB5 NERT cells following the addition of OHT (Fig. 4, 5, 6, 7, Table S1). As expected, Hes and Hey genes showed a similar or even higher induction of mRNA expression by activated Notch1 in the presence of CHX (Fig. 4, 5, 6, 7, Table S1), confirming that Hes and Hey genes are direct targets of Notch signaling. Interestingly, the well known Notch target genes Hes1 and Hes7 were also cell specifically induced by activated Notch1: In the absence of CHX, the expression of Hes1 was only up- regulated by activated Notch1 in ESC but not in mesodermal cells, while Hes7 was up-regulated only in mesodermal cells but not in ESC (Fig. 4, 5, 6, 7 and Table S1). Taken together, we conclude that Notch1 exerts its multiple effects by specifically regulating the expression of genes in a highly cell intrinsic and cell extrinsic context dependent manner. Notch1/RBP-J signaling induces expression of key transcriptional regulators involved in cell lineage determination in the absence of protein synthesis The major function of Notch signaling is to regulate differentiation decisions. Classically, this pathway is employed to restrict cell differentiation by signaling through the Hes/Hey transcriptional repressors. In this study however, we found that a number of further key regulatory transcription factors essential for lineage determination and cell differentiation were induced by Notch1 signaling in a cell context dependent manner (Figs. 4, 6, 7). These included transcription factors that play a role in all three germ layers, such as Sox9, transcription factors that are essential specifically in neuro-ectodermal development, such as Pax6, and transcription factors required for lineage specification of mesoder- mal derived tissues, such as Myf5 for skeletal muscle development and Runx1 for hematopoietic stem cell fate of mesodermal progenitor cells. Furthermore, Notch1/RBP-J signaling differen- tially regulated expression of members of the Id family of transcriptional repressors that interfere with the transcriptional activities of differentiation inducing transcription factors. Impor- tantly, all of these genes were also induced by activated Notch1 to a similar or even higher extent in the absence of protein synthesis (Fig. 7, 8), suggesting that they represent direct target genes of Notch1. To further analyze a potential direct regulation, the promoter sequences of these genes were screened for RBP-J binding site sequences. As shown in Figure 9, the promoters of these transcription factors contained consensus RBP-J binding sites, thus supporting a model by which Notch signaling influences lineage decisions by directly activating key transcriptional regulators involved in cell lineage determination (see model below). The transcription factor Sox9 is a primary Notch1 target gene Among the genes that were induced by activated Notch1 in undifferentiated ESC (ESCs), in ESC differentiating along the ectodermal (ESCe) or mesodermal (ESCm) lineages as well as in mesodermal cells (Mesoderm) was Sox9 (Fig. 4, 7, 8D), an essential transcription factor involved in development of all three germ layers. In line with our RNA data, activated Notch1 shifted Sox9 protein expression from undetectable to an intermediate level in ESCs (Fig. 10A) and in 5 d differentiated embryoid bodies from an intermediate to high level expression (Fig. 10A), confirming a valid role of Notch for Sox9 regulation in both undifferentiated and differentiated cells. Interestingly, RBP-J has been shown to be required to maintain Sox9 expression during the gliogenic phase of spinal cord development [20] and Notch signaling is essential for Sox9 expression in the mouse retina [21], however whether Notch signaling directly or indirectly regulates Sox9 expression has not been determined. Here we show that Sox9 is upregulated by activated Notch1 even when protein synthesis is inhibited by CHX PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11481