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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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Most unique reads map within 1.5 kb of the transcriptional start site.Uniquely-aligned sequences (reads) were counted within a given 1000 base window relative to genomic positions. A step size of 50 bases was used for window overlap. Poisson distribution [116] was then used to generate P-values for each 1000 base window and windows were filtered by these values to generate a list of peaks. Significant peaks were windows with P-value less than 10−12.
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pone-0020022-g001: Most unique reads map within 1.5 kb of the transcriptional start site.Uniquely-aligned sequences (reads) were counted within a given 1000 base window relative to genomic positions. A step size of 50 bases was used for window overlap. Poisson distribution [116] was then used to generate P-values for each 1000 base window and windows were filtered by these values to generate a list of peaks. Significant peaks were windows with P-value less than 10−12.

Mentions: These ChIP purified DNA fragments were then sequenced using massive parallel sequencing instead of hydbridizing them to an array. An experiment sequenced in technical triplicates resulted in 36 base pair (bp) sequence reads which were aligned to the reference mouse genome. 72.9% of the Notch On and 83.9% of the Notch Off uniquely mapped reads aligned to the genome with zero-mismatches. Figure 1 shows that the unique reads mapped preferentially to regions within 1.5 kb from the transcriptional start sites indicating that the Re-ChIP DNA fragments are enriched in the proximal promoter of genes instead of randomly distributed.


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

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

Most unique reads map within 1.5 kb of the transcriptional start site.Uniquely-aligned sequences (reads) were counted within a given 1000 base window relative to genomic positions. A step size of 50 bases was used for window overlap. Poisson distribution [116] was then used to generate P-values for each 1000 base window and windows were filtered by these values to generate a list of peaks. Significant peaks were windows with P-value less than 10−12.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020022-g001: Most unique reads map within 1.5 kb of the transcriptional start site.Uniquely-aligned sequences (reads) were counted within a given 1000 base window relative to genomic positions. A step size of 50 bases was used for window overlap. Poisson distribution [116] was then used to generate P-values for each 1000 base window and windows were filtered by these values to generate a list of peaks. Significant peaks were windows with P-value less than 10−12.
Mentions: These ChIP purified DNA fragments were then sequenced using massive parallel sequencing instead of hydbridizing them to an array. An experiment sequenced in technical triplicates resulted in 36 base pair (bp) sequence reads which were aligned to the reference mouse genome. 72.9% of the Notch On and 83.9% of the Notch Off uniquely mapped reads aligned to the genome with zero-mismatches. Figure 1 shows that the unique reads mapped preferentially to regions within 1.5 kb from the transcriptional start sites indicating that the Re-ChIP DNA fragments are enriched in the proximal promoter of genes instead of randomly distributed.

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