Physiologically, Notch signal transduction plays a pivotal role in differentiation; pathologically, Notch signaling contributes to the development of cancer. Transcriptional activation of Notch target genes involves cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), and NICD migration into the nucleus and assembly of a coactivator complex. Posttranslational modifications of the NICD are important for its transcriptional activity and protein turnover. Deregulation of Notch signaling and stabilizing mutations of Notch1 have been linked to leukemia development. We found that the methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1; also known as PRMT4) methylated NICD at five conserved arginine residues within the C-terminal transactivation domain. CARM1 physically and functionally interacted with the NICD-coactivator complex and was found at gene enhancers in a Notch-dependent manner. Although a methylation-defective NICD mutant was biochemically more stable, this mutant was biologically less active as measured with Notch assays in embryos of Xenopus laevis and Danio rerio. Mathematical modeling indicated that full but short and transient Notch signaling required methylation of NICD.
Site-specific methylation of Notch1 controls the amplitude and duration of the Notch1 response.
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Notch1 signaling is absolutely essential for steady-state thymic lymphopoiesis, but the role of other Notch receptors, and their potential overlap with the function of Notch1, remains unclear. Here we show that like Notch1, Notch3 is differentially expressed by progenitor thymocytes, peaking at the DN3 progenitor stage. Using mice carrying a gene-trapped allele, we show that thymic cellularity is slightly reduced in the absence of Notch3, although progression through the defined sequence of TCR- development is normal, as are NKT and TCR cell production.
Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis.
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Notch1-IC, Notch2-IC or EBNA2 have been induced in a conditionally immortalized human B cell line (EREB2-5) in order to identify similar and unique target genes in B cells. CAT was used as a control.
Notch1, Notch2, and Epstein-Barr virus-encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus-infected B cells.
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Airway basal cells (BC) function as progenitor cells capable of differentiating into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with -secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secre-tory cells and ciliated cells, but more so for the secretory lineage. Sustained Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the Notch 2 and 4 pathways have little effect on BC differentiation, while activation of the Notch1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment.
Activation of NOTCH1 or NOTCH3 signaling skews human airway basal cell differentiation toward a secretory pathway.
Specimen part, TimeView Samples
Notch3 is a transmembrane receptor which is critically important for the structure and myogenic response of distal arteries, particularly cerebral arteries. After activation of the receptor, the intracellular domain translocates in the nucleus to activate target genes transcription.
Transcriptome analysis for Notch3 target genes identifies Grip2 as a novel regulator of myogenic response in the cerebrovasculature.
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Purpose: To use RNA-Seq analysis of endothelial cell in various Notch1 alllels in order to determine transcrptional differencesas a consequence of Notch dose. Methods: Using a FACS sorting we generated high-throughput RNA-SEQ data of endothelials in various Notch1 alleles during development Results: Notch1 dose can alter gene expression in a subset of endothelial genes Overall design: RNA-Seq was performed on endothelial cells isolated at e9.5 from embryos with various Notch1 alleles including N1+/+, N1+/-, N1+/vg, N112/vg, N112/-
The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis.
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Ozone is a highly toxic air pollutant and global health concern. Mechanisms of genetic susceptibility to ozone-induced lung inflammation are not completely understood. We hypothesized Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation. Wild type (WT), Notch3 (Notch3-/-) and Notch4 (Notch4-/-) knockout mice were exposed to ozone (0.3 ppm) or filtered air for 6-72 hours. Ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared to WT and Notch3-/-. Significantly greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared to WT mice after ozone. Expression of whole lung Tnf was significantly increased after ozone in all genotypes, and was significantly greater in Notch3-/- mice compared to WT. Statistical analyses of the transcriptome identified differentially expressed gene networks between WT and knockout mice basally and after ozone, and included Trim30, a member of the inflammasome pathway, and Traf6, an inflammatory signaling member. These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation.
Novel Roles for Notch3 and Notch4 Receptors in Gene Expression and Susceptibility to Ozone-Induced Lung Inflammation in Mice.
Specimen partView Samples
Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype.
Notch signaling functions in retinal pericyte survival.
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Background: Interaction between key signaling mechanisms is important to generate the diversity in signaling output required for proper control of cellular differentiation and function, although the molecular manifestations of such cross-talk are only partially understood. Notch signaling and the cellular response to hypoxia intersect at different points in the signaling cascades, and in this report we analyze the consequences of this cross-talk at the transcriptome level. Results: Mouse ES cells were subjected to various combinations of hypoxia and/or activated Notch signaling, and the transcriptome changes could be grouped into different categories, reflecting various modes of hypoxia and Notch signaling integration. Two principal categories of novel Notch- and hypoxia-induced genes were identified: i) a larger set of genes induced by one pathway and not significantly affected by the activity status of the other pathway; and ii) a smaller set of genes co-regulated by Notch and hypoxia. In the latter category, we identified genes that were induced by hypoxia and the expression of which was enhanced by active Notch signaling. In addition, a number of genes were induced by Notch and hypoxia independently, and a final category of genes required simultaneous activation of Notch and hypoxia to be significantly induced. Several of the hypoxia- and Notch-induced genes were found to be upregulated in various forms of cancer. Conclusions: We identify novel Notch and hypoxia downstream genes and genes co-regulated by the two pathways, providing a molecular platform to better understand the intersection between the two signaling cascades in normal development and cancer.
Interactions between Notch- and hypoxia-induced transcriptomes in embryonic stem cells.
Sex, Specimen part, TreatmentView Samples
Ascending aortic aneurysms (AscAA) are a life-threatening disease whose molecular basis is poorly understood. Mutations in NOTCH1 have been linked to bicuspid aortic valve (BAV), which is associated with AscAA. Here, we describe a novel role for Notch1 in AscAA. We found that Notch1 haploinsufficiency exacerbated the aneurysmal aortic root dilation seen in the Marfan syndrome mouse model and that heterozygous deletion of Notch1 in the second heart field (SHF) lineage recapitulated this exacerbated phenotype. Lineage tracing analysis showed that loss of Notch1 in the SHF reduces the number of SHF-derived smooth muscle cells in the aortic root, and RNA-seq analysis demonstrated distinct in vivo expression patterns between lineage-specific regions of the ascending aorta. Finally, Notch1+/- mice in a predominantly 129S6 background develop aortic root dilation, indicating that loss of Notch1 independently predisposes to AscAA. These findings are the first to demonstrate a SHF lineage-specific role for Notch1 in AscAA and suggest that genes linked to the development of BAV may also contribute to the associated aortopathy. Overall design: To determine why dilation was localized to the aortic root in Notch1.129S6+/- mice, RNA-sequencing was performed on proximal and distal ascending aortic tissue from Notch1.129S6+/- mice and wildtype littermates at 2 months of age. Transcriptome analysis was utilized to better understand why the dilation was localized to the aortic root. Hierarchical cluster analysis grouped these samples based on location first and then genotype, and showed that cells of the proximal and distal ascending aorta have distinct gene expression patterns in vivo.
Notch1 haploinsufficiency causes ascending aortic aneurysms in mice.
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