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Identification of novel targets of CSL-dependent Notch signaling in hematopoiesis.

Hamidi H, Gustafason D, Pellegrini M, Gasson J - PLoS ONE (2011)

Bottom Line: Notch 1 does not bind DNA but instead binds the CSL transcription factor to regulate gene expression.Finally, 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of in vitro hematopoiesis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

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.

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MicroRNA differentially regulated from Day 5 to Day 8.Dendrogram showing a) the 83 differentially regulated microRNAs from day 5 to day 8. b) microRNAs that were differentially regulated comparing Day 8 Notch ON to Notch OFF and c) the microRNAs used for real-time PCR analysis. Data analyses were performed by using DNA-Chip Analyzer 1.3 [117]. The thresholds for selecting significant genes were set at a relative difference of >1.5-fold, an absolute difference of >100 signal intensity, and P<0.05.
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pone-0020022-g004: MicroRNA differentially regulated from Day 5 to Day 8.Dendrogram showing a) the 83 differentially regulated microRNAs from day 5 to day 8. b) microRNAs that were differentially regulated comparing Day 8 Notch ON to Notch OFF and c) the microRNAs used for real-time PCR analysis. Data analyses were performed by using DNA-Chip Analyzer 1.3 [117]. The thresholds for selecting significant genes were set at a relative difference of >1.5-fold, an absolute difference of >100 signal intensity, and P<0.05.

Mentions: MicroRNA are important regulator of hematopoiesis and we wanted to assess which microRNA are differentially expressed during hematopoiesis in our in vitro murine embryonic stem cell co-culture system. Expression profiles of miRNA during normal hematopoietic differention were examined using a commercial miRNA microarray that contained all miRNA present in the miRBase miRNA registry release 8.1 [98], [99]. To assess if miRNA are differentially regulated during normal hematopoiesis, the expression profile of miRNA from hematopoietic progenitors (day 8) were compared to flk1+ hemangioblasts (day 5). Each time point was represented by two independent biological samples and two technical replicates. Microarray analysis revealed that 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of hematopoiesis in vitro from day 5 to day 8 (table S1). Thirty one miRNA were up-regulated and fifty two were down-regulated. The differentially regulated miRNA cluster into groups (see figure 4a).


Identification of novel targets of CSL-dependent Notch signaling in hematopoiesis.

Hamidi H, Gustafason D, Pellegrini M, Gasson J - PLoS ONE (2011)

MicroRNA differentially regulated from Day 5 to Day 8.Dendrogram showing a) the 83 differentially regulated microRNAs from day 5 to day 8. b) microRNAs that were differentially regulated comparing Day 8 Notch ON to Notch OFF and c) the microRNAs used for real-time PCR analysis. Data analyses were performed by using DNA-Chip Analyzer 1.3 [117]. The thresholds for selecting significant genes were set at a relative difference of >1.5-fold, an absolute difference of >100 signal intensity, and P<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102675&req=5

pone-0020022-g004: MicroRNA differentially regulated from Day 5 to Day 8.Dendrogram showing a) the 83 differentially regulated microRNAs from day 5 to day 8. b) microRNAs that were differentially regulated comparing Day 8 Notch ON to Notch OFF and c) the microRNAs used for real-time PCR analysis. Data analyses were performed by using DNA-Chip Analyzer 1.3 [117]. The thresholds for selecting significant genes were set at a relative difference of >1.5-fold, an absolute difference of >100 signal intensity, and P<0.05.
Mentions: MicroRNA are important regulator of hematopoiesis and we wanted to assess which microRNA are differentially expressed during hematopoiesis in our in vitro murine embryonic stem cell co-culture system. Expression profiles of miRNA during normal hematopoietic differention were examined using a commercial miRNA microarray that contained all miRNA present in the miRBase miRNA registry release 8.1 [98], [99]. To assess if miRNA are differentially regulated during normal hematopoiesis, the expression profile of miRNA from hematopoietic progenitors (day 8) were compared to flk1+ hemangioblasts (day 5). Each time point was represented by two independent biological samples and two technical replicates. Microarray analysis revealed that 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of hematopoiesis in vitro from day 5 to day 8 (table S1). Thirty one miRNA were up-regulated and fifty two were down-regulated. The differentially regulated miRNA cluster into groups (see figure 4a).

Bottom Line: Notch 1 does not bind DNA but instead binds the CSL transcription factor to regulate gene expression.Finally, 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of in vitro hematopoiesis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus