title: Identification of novel targets of CSL-dependent Notch signaling in hematopoiesis.
abstract: Somatic activating mutations in the Notch1 receptor result in the overexpression of activated Notch1, which can be tumorigenic. The goal of this study is to understand the molecular mechanisms underlying the phenotypic changes caused by the overexpression of ligand independent Notch 1 by using a tetracycline inducible promoter in an in vitro embryonic stem (ES) cells/OP9 stromal cells coculture system, recapitulating normal hematopoiesis. First, an in silico analysis of the promoters of Notch regulated genes (previously determined by microarray analysis) revealed that the motifs recognized by regulatory proteins known to mediate hematopoiesis were overrepresented. Notch 1 does not bind DNA but instead binds the CSL transcription factor to regulate gene expression. The in silico analysis also showed that there were putative CSL binding sites observed in the promoters of 28 out of 148 genes. A custom ChIP-chip array was used to assess the occupancy of CSL in the promoter regions of the Notch1 regulated genes in vivo and showed that 61 genes were bound by activated Notch responsive CSL. Then, comprehensive mapping of the CSL binding sites genome-wide using ChIP-seq analysis revealed that over 10,000 genes were bound within 10 kb of the TSS (transcription start site). The majority of the targets discovered by ChIP-seq belong to pathways that have been shown by others to crosstalk with Notch signaling. Finally, 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of in vitro hematopoiesis. Thirty one miRNA were up-regulated and fifty two were down-regulated. Overexpression of Notch1 altered this pattern of expression of microRNA: six miRNAs were up-regulated and four were down regulated as a result of activated Notch1 overexpression during the course of hematopoiesis. Time course analysis of hematopoietic development revealed that cells with Notch 1 overexpression mimic miRNA expression of cells in a less mature stage, which is consistent with our previous biological characterization.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Habib
authors.initials: H
authors.lastname: Hamidi
authors.forename: Derek
authors.initials: D
authors.lastname: Gustafason
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.forename: Judith
authors.initials: J
authors.lastname: Gasson
mesh_terms: D000818:Animals; D020029:Base Pairing / Q000235:genetics; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D056426:Genetic Loci / Q000235:genetics; D016678:Genome / Q000235:genetics; D006410:Hematopoiesis / Q000235:genetics*; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D035683:MicroRNAs; C497571:Rbpj protein, mouse; D051880:Receptors, Notch