Effect of Network-Correcting Molecules on NOTCH1 (N1)-Haploinsufficient Human Induced Pluripotent Stem Cell (iPSC)-Derived Endothelial Cells (ECs)
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ABSTRACT:
PROVIDER: PRJNA515762 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8453500_1.fastq.gz | Fastqsanger.gz | |||
| SRR8453500_2.fastq.gz | Fastqsanger.gz | |||
| SRR8453510_1.fastq.gz | Fastqsanger.gz | |||
| SRR8453510_2.fastq.gz | Fastqsanger.gz | |||
| SRR8453520_1.fastq.gz | Fastqsanger.gz |
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Project description: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 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/-
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Project description:NOTCH1 (N1) is a transmembrane receptor that initiates a cell-cell signaling pathway controlling various cell fate specifications in metazoans. The addition of O-fucose by Protein O-fucosyltransferase 1 (POFUT1) to Epidermal Growth Factor-like (EGF) repeats in the N1 extracellular domain is essential for N1 function, and modification of O-fucose with GlcNAc by the Fringe family of glycosyltransferases modulates Notch activity. Prior cell-based studies showed that POFUT1 modifies EGF repeats containing the appropriate consensus sequence at high stoichiometry, while Fringe GlcNAc-transferases (LFNG, MFNG and RFNG) modify O-fucose on only a subset of NOTCH1 EGF repeats. Previous in vivo studies showed that each FNG affects naïve T cell development. To examine Fringe modifications of N1 expressed at a physiological level, we used mass spectral glycoproteomic methods to analyze O-fucose glycans of endogenous N1 from activated T cells obtained from mice lacking all Fringe enzymes, or expressing only a single FNG. While most O-fucose sites were modified at high stoichiometry, only EGF6, EGF16, EGF26, and EGF27 were extended in control T cells. Cell-based assays of N1 lacking fucose at each of those O-fucose sites revealed minor functional correlations in the EGF16 and EGF27 mutant with Notch ligand binding. In activated T cells expressing only LFNG, MFNG or RFNG alone, the extension of O-fucose with GlcNAc in the same EGF repeats was diminished, consistent with cooperative interactions when all three Fringes were present. The combined data open the door for the analysis of O-glycans on endogenous N1 derived from different cell types.
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Project description:Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG) and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG.
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Project description:Notch signaling is a core patterning module for vascular morphogenesis, which co-determines the sprouting behaviour of endothelial cells (ECs). Tight quantitative and temporal control of Notch activity is essential for vascular development, yet the details of Notch regulation in ECs are incompletely understood. We found that ubiquitin-specific peptidase 10 (USP10) interacted with the NOTCH1 intracellular domain (NICD1) to slow the ubiquitin-dependent turnover of this short-lived form of the activated NOTCH1 receptor. Accordingly, inactivation of USP10 reduced NICD1 abundance and stability, and diminished Notch-induced target gene expression in ECs. In mice, loss of endothelial Usp10 increased vessel sprouting and partially restored the patterning defects caused by ectopic expression of NICD1. Thus, USP10 functions as an NICD1 deubiquitinase, which fine-tunes endothelial Notch responses during angiogenic sprouting.
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Project description:NOTCH1 (N1) is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH extracellular domain, which is required for signaling activation and trafficking. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of N1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on N1 in POFUT1 S162L patient fibroblasts is the cause of these effects, we immunopurified endogenous N1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. N1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of N1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 has significant effects on N1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.
2024-08-02 | PXD050256 | Pride
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