Modulation Of Notch Signaling Research Articles

Notch Signaling and MicroRNA: The Dynamic Duo Steering Between Neurogenesis and Glioblastomas.

Notch signaling is an evolutionary conserved pathway that plays a central role in development and differentiation of eukaryotic cells. It has been well documented that Notch signaling is inevitable for neuronal cell growth and homeostasis. It regulates process of differentiation from early embryonic stages to fully developed brain. To achieve this streamlined development of neuronal cells, a number of cellular processes are being orchestrated by the Notch signaling. Abrogated Notch signaling is related to several brain tumors, including glioblastomas. On the other hand, microRNAs are small molecules that play decisive role in mediating and modulating Notch signaling. This review discusses the crucial role of Notch signaling in development of nervous system and how this versatile pathway interplay with microRNAs in glioblastoma. This review sheds light on interplay between abrogated Notch signaling and miRNAs in the regulation of neuronal differentiation with special focus on miRNAs mediated regulation of tumorigenesis in glioblastoma. Furthermore, it discusses different aspects of neurogenesis modulated by the Notch signaling that could be exploited for the identification of new diagnostic tools and therapies for the treatment of glioblastoma.

The exocyst complex regulates C. elegans germline stem cell proliferation by controlling membrane Notch levels.

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels.

Notoginsenoside R1 activates the NAMPT-NAD+-SIRT1 cascade to promote postischemic angiogenesis by modulating Notch signaling

Nicotinamide phosphoribosyltransferase (NAMPT) maintains mitochondrial function and protects against cerebral ischemic injury by improving energy metabolism. Notoginsenoside R1 (R1), a unique constituent of Panax notoginseng, has been shown to promote the proliferation and tube formation of human umbilical vein endothelial cells. Whether R1 has proangiogenesis on the activation of NAMPT in ischemic stroke remains unclear. The purpose of this study was to investigate the pharmacodynamic effect and mechanism of R1 on angiogenesis after ischemic stroke. We used male Sprague-Dawley (SD) rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered via intraperitoneal (i.p.) injection immediately after ischemia induction. The promotion of R1 on angiogenesis were detected by immunofluorescence staining, 3D stereoscopic imaging and transmission electron microscopy detection. HBMEC cells were pretreated with different concentrations of R1 for 12 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, scratch assay, EdU staining and tube formation were determined. Western blot analyses of proteins, including those involved in angiogenesis, NAMPT-SIRT1 cascade, VEGFR-2, and Notch signaling, were conducted. We showed that R1 significantly restored cerebral blood flow, improved mitochondrial energy metabolism and promoted angiogenesis. More importantly, incubation with 12.5–50 μM R1 significantly increased the migration, proliferation and tube formation of HBMECs in vitro. The promotion of R1 on angiogenesis were associated with the NAMPT-NAD+-SIRT1 cascade and Notch/VEGFR-2 signaling pathway, which was partially eliminated by inhibitors of NAMPT and SIRT1. We demonstrated that R1 promotes post-stroke angiogenesis via activating NAMPT-NAD+-SIRT1 cascade. The modulation of Notch signaling and VEGFR-2 contribute to the post-stroke angiogenesis. These findings offer insight for exploring new therapeutic strategies for neurorestoration via R1 treatment after ischemic stroke.

Ogt controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling

Ogt catalyzed O-linked N-acetylglucosamine (O-GlcNAcylation, O-GlcNAc) plays an important function in diverse biological processes and diseases. However, the roles of Ogt in regulating neurogenesis remain largely unknown. Here, we show that Ogt deficiency or depletion in adult neural stem/progenitor cells (aNSPCs) leads to the diminishment of the aNSPC pool and aberrant neurogenesis and consequently impairs cognitive function in adult mice. RNA sequencing reveals that Ogt deficiency alters the transcription of genes relating to cell cycle, neurogenesis, and neuronal development. Mechanistic studies show that Ogt directly interacts with Notch1 and catalyzes the O-GlcNAc modification of Notch TM/ICD fragment. Decreased O-GlcNAc modification of TM/ICD increases the binding of E3 ubiquitin ligase Itch to TM/ICD and promotes its degradation. Itch knockdown rescues neurogenic defects induced by Ogt deficiency invitro and invivo. Our findings reveal the essential roles and mechanisms of Ogt and O-GlcNAc modification in regulating mammalian neurogenesis and cognition.

Characterization of a new mastermind allele identified from somatic mosaic screen.

The Notch signaling pathway is highly conserved and regulates various fundamental development events. Activation of Notch signaling relies on production of the Notch intracellular domain (NICD), which assembles a transcription factor complex to turn on down-stream targets expression. The mastermind (mam) gene encodes an essential co-activator that permits NICD activity in the cell nucleus. During a somatic mosaic screen in Drosophila, an uncharacterized gene l(2)S9998 is identified as a positive regulator of the Notch signaling pathway. Genetic analysis demonstrates that l(2)S9998 functions at the level of transcriptional activation of Notch targets in the signal receiving cells. Whole genome sequencing reveals that l(2)S9998 is a novel allele of the mam gene, which is further confirmed by complementation tests. Along with three molecularly defined transposon insertions isolated from the screen, four mutants of mam are shown to modulate Notch signaling during fly wing development. Our analysis provides additional genetic resources for understanding mam function and Notch signaling regulation.

Dual-Specificity Phosphatase 15 (DUSP15) Modulates Notch Signaling by Enhancing the Stability of Notch Protein.

Dual-specificity phosphatases (DUSPs) comprise a unique group of enzymes that dephosphorylate signaling proteins at both phospho-serine/threonine and phospho-tyrosine residues. Since Notch signaling is an essential pathway for neuronal cell fate determination and development that is also upregulated in Alzheimer's disease tissues, we sought to explore whether and how DUSPs may impact Notch processing. Our results show that overexpression of DUSP15 concomitantly and dose-dependently increased the steady-state levels of recombinant Notch (extracellular domain-truncated Notch, NotchΔE) protein and its cleaved product, Notch intracellular domain (NICD). The overall ratio of NotchΔE to NICD was unchanged by overexpression of DUSP15, suggesting that the effect is independent of γ-secretase. Interestingly, overexpression of DUSP15 also dose-dependently increased phosphorylated ERK1/2. Phosphorylated ERK1/2 is known to be positively correlated with Notch protein level, and we found that DUSP15-mediated regulation of Notch was dependent on ERK1/2 activity. Together, our findings reveal the existence of a previously unidentified DUSP15-ERK1/2-Notch signaling axis, which could potentially play a role in neuronal differentiation and neurological disease.

Inhibition of gamma-secretase activity without interfering in Notch signalling decreases inflammatory response in patients with cutaneous leishmaniasis

ABSTRACT Cutaneous leishmaniasis (CL) patients present an exacerbated inflammatory response associated with tissue damage and ulcer development. Increasing numbers of patients have exhibited treatment failure, which remains not well understood. We hypothesized that adjuvant anti-inflammatory therapy would benefit CL patients. The aim of the present study was to investigate the contribution of Notch signalling and gamma-secretase activity to the inflammatory response observed in CL patients. Notch signalling is a molecular signalling pathway conserved among animal species. Gamma-secretase forms a complex of proteins that, among other pathways, modulates Notch signalling and immune response. We found that Notch 1 cell receptor signalling protects against the pathologic inflammatory response, and JLK6, a gamma-secretase inhibitor that does not interfere with Notch signalling, was shown to decrease the in-vitro inflammatory response in CL. Our data suggest that JLK6 may serve as an adjuvant treatment for CL patients.

Designing Biomaterials to Modulate Notch Signaling in Tissue Engineering and Regenerative Medicine.

The design of cell-instructive biomaterials for tissue engineering and regenerative medicine is at a crossroads. Although the conventional tissue engineering approach is top-down (cells seeded to macroporous scaffolds and mature to form tissues), bottom-up tissue engineering strategies are becoming appealing. With such developments, we can study cell signaling events, thus enabling functional tissue assembly in physiologic and diseased models. Among many important signaling pathways, the Notch signaling pathway is the most diverse in its influence during tissue morphogenesis and repair following injury. Although Notch signaling is extensively studied in developmental biology and cancer biology, our knowledge of designing biomaterial-based Notch signaling platforms and incorporating Notch signaling components into engineered tissue systems is limited. By incorporating Notch signaling to tissue engineering scaffolds, we can direct cell-specific responses and improve engineered tissue maturation. This review will discuss recent progress in the development of Notch signaling biomaterials as a promising target to control cellular fate decisions, including the influences of ligand identity, biophysical material cues, ligand presentation strategies, and mechanotransduction. Notch signaling is consequently of interest to direct, control, and reprogram cellular behavior on a biomaterial surface. We anticipate that discussions in this article will allow for enhanced knowledge and insight into designing Notch targeted biomaterials for various tissue engineering and cell fate determinations. Impact statement Notch signaling is recognized as an important pathway in tissue engineering and regenerative medicine; however, there is no systematic review on this topic. The comprehensive review and perspectives presented here provide an in-depth discussion on ligand presentation strategies both in 2D and in 3D cell culture environments involving biomaterials/scaffolds. In addition, this review article provides insight into the challenges in designing cell surrogate biomaterials capable of providing Notch signals. To the best of the authors' knowledge, this is the first review relevant to the fields of tissue engineering.

Haematopoietic stem cell-dependent Notch transcription is mediated by p53 through the Histone chaperone Supt16h.

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.

SOX2/SALL4 stemness axis modulates Notch signaling genes to maintain self-renewal capacity of esophageal squamous cell carcinoma.

Stemness phenotype is considered as the centerpiece of cancer biology due to its potential in conventional chemo-radiotherapy resistance and tumor recurrence after clinical intervention. This feature in tumor mass belongs to activation of core regulatory stemness factors and different cell signaling pathways in cancer stem cells. We aimed in this study to elucidate contribution of Notch signaling pathway in stemness state of esophageal squamous cell carcinoma (ESCC) through their relevance with stem cell markers SOX2 and SALL4. 50 ESCC tumor and related margin normal tissues were considered and categorized based on SOX2/SALL4 expression pattern, and mRNA levels of Notch signaling genes including ligands, receptors, target genes, and transcriptional coactivator were analyzed in the selected groups using qRT-PCR. Concomitant overexpression of stem cell markers SOX2 and SALL4 in ESCCs upregulated the involved genes in Notch signaling pathway. Upregulation of Notch pathway genes associated with depth of tumor invasion and lymph node metastasis of ESCC. Based on biological function of SOX2 and SALL4 axis in stemness state potential, our results may suggest contribution of Notch signaling pathway in self-renewal capacity of ESCCs, as well as invasion and metastasis of the disease. To the best of our knowledge, this is the first report elucidating the crosstalk between SOX2/SALL4 stemness factors and Notch signaling pathway in cancer research.

Notch signaling and the emergence of hematopoietic stem cells.

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.

LncRNA RUSC1-AS1 promotes the proliferation of hepatocellular carcinoma cells through modulating NOTCH signaling.

The long non-coding RNA (lncRNA) RUSC1-AS1 has been reported to be dysregulated in the progression of many cancers. Also, RUSC1-AS1 had been detected to be highly expressed in laryngeal squamous cell carcinoma and breast cancer cells, suggesting that RUSC1-AS1 may be a biomarker for cancers. However, the biological role and regulatory mechanism of RUSC1-AS1 in hepatocellular carcinoma (HCC) remain unknown. In this study, we found that RUSC1-AS1 was upregulated in HCC tissues and cells, and predicted unfavorable prognosis of HCC patients. The function assays including colony formation, EdU, TUNEL assay revealed that RUSC1-AS1 facilitated HCC cell proliferation and inhibited HCC cell apoptosis. Furthermore, mechanism assays including luciferase reporter assay and RIP assay demonstrated that RUSC1-AS1 could directly bind to hsa-miR-7-5p. Besides, hsa-miR-7-5p targeted and negatively regulated NOTCH3 expression. Moreover, RUSC1-AS1 sponged hsa-miR-7-5p to upregulate NOTCH3 and to trigger the NOTCH signaling pathway. The rescue assays depicted that RUSC1-AS1 regulated HCC cell proliferation and apoptosis through modulating NOTCH signaling. In conclusion, lncRNA RUSC1-AS1 promoted the proliferation and reduced the apoptosis of HCC cells through activation of NOTCH signaling via hsa-miR-7-5p/NOTCH3 axis.

Epidermal Growth Factor-Like Domain-8 (EGFL8) in Mammals: The Story so far


 
 
 Epidermal growth factor-like domain-8 (EGFL8) also known as vascular endothelial statin-2 (VE-statin-2). It was identified by using retroviral gene entrapment vectors, expressed in endothelial cells. It is located on chromosome6inhumansandchromosome17inthemouse. EGFL8codesaproteinof293aminoacidswithan amino-terminal signal peptide and has two EGF-like domains. EGFL8 plays a very important regulatory role in thymopoiesis, cell migration and invasion through the modulation of Notch signaling. Although the signaling regulatory factors of EGFL8 need to be explored but recent scientific advances have revealed some important aspect of its regulation. This review summarizes the current knowledge about all aspects of EGFL8 since its discovery.
 
 

Multifaceted regulation of Notch signaling by glycosylation.

To build a complex body composed of various cell types and tissues and to maintain tissue homeostasis in the postembryonic period, animals use a small number of highly conserved intercellular communication pathways. Among these is the Notch signaling pathway, which is mediated via the interaction of transmembrane Notch receptors and ligands usually expressed by neighboring cells. Maintaining optimal Notch pathway activity is essential for normal development, as evidenced by various human diseases caused by decreased and increased Notch signaling. It is therefore not surprising that multiple mechanisms are used to control the activation of this pathway in time and space. Over the last 20years, protein glycosylation has been recognized as a major regulatory mechanism for Notch signaling. In this review, we will provide a summary of the various types of glycan that have been shown to modulate Notch signaling. Building on recent advances in the biochemistry, structural biology, cell biology and genetics of Notch receptors and the glycosyltransferases that modify them, we will provide a detailed discussion on how various steps during Notch activation are regulated by glycans. Our hope is that the current review article will stimulate additional research in the field of Notch glycobiology and will potentially be of benefit to investigators examining the contribution of glycosylation to other developmental processes.

Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells

Gene regulation and metabolism are two fundamental processes that coordinate the self-renewal and differentiation of neural precursor cells (NPCs) in the developing mammalian brain. However, little is known about how metabolic signals instruct gene expression to control NPC homeostasis. Here, we show that methylglyoxal, a glycolytic intermediate metabolite, modulates Notch signalling to regulate NPC fate decision. We find that increased methylglyoxal suppresses the translation of Notch1 receptor mRNA in mouse and human NPCs, which is mediated by binding of the glycolytic enzyme GAPDH to an AU-rich region within Notch1 3ʹUTR. Interestingly, methylglyoxal inhibits the enzymatic activity of GAPDH and engages it as an RNA-binding protein to suppress Notch1 translation. Reducing GAPDH levels or restoring Notch signalling rescues methylglyoxal-induced NPC depletion and premature differentiation in the developing mouse cortex. Taken together, our data indicates that methylglyoxal couples the metabolic and translational control of Notch signalling to control NPC homeostasis.

Shaping of the Tumor Microenvironment by Notch Signaling.

The tumor microenvironment (TME) has become a major concern of cancer research both from a basic and a therapeutic point of view. Understanding the effect of a signaling pathway-and thus the effect of its targeting-in every aspect of the microenvironment is a prerequisite to predict and analyze the effect of a therapy. The Notch signaling pathway is involved in every component of the TME as well as in the interaction between the different parts of the TME. This review aims at describing how Notch signaling is impacting the TME and the consequences this may have when modulating Notch signaling in a therapeutic perspective.

Interrogation of Individual CLL Loss-of-Function Lesions By CRISPR In Vivo Editing Reveals Common and Unique Pathway Alterations

Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a transplant model that can rapidly evaluate genetic lesions. First, we crossed mice carrying B-cell restricted Cre expression (Cd19-cre) with mice carrying conditional Cas9-GFP, to generate a strain expressing B cell-restricted Cas9 (Cd19-Cas9). Next, we optimized methods for in vitro engineering of early stem and progenitor cells (Lin- Sca-1+ c-kit+ [LSK]) from Cd19-Cas9 mice using lentivirus expressing sgRNAs (mCherry+)targeting Atm, Trp53, Chd2, Birc3, Mga, or Samhd1. We chose LSKs because of their high transducibility and long-term repopulating potential. Last, we transplanted the single sgRNA-expressing LSKs into sub-lethally irradiated CD45.1 recipient mice, and then confirmed presence of ~45-85% gene-edited sequences (>70% carrying frameshift mutations) in edited B cells (GFP+mCherry+) at 2 months post-transplant, by PCR-based targeted deep sequencing and CRISPResso software analysis. We also verified presence of gene alterations (and putative off-target lesions) at the single cell DNA level (targeted sequencing by Tapestri, Mission Bio). We first asked whether presence of the 6 LOFs could impact B cell developmental trajectories in marrow, spleen and peritoneum at 4 months post-transplant, a time point by which B cells are considered to achieve optimal host reconstitution (n=5/group, including a non-targeting control group). No marked changes were observed in mice with Atmindel, Trp53indel, Chd2indel, Birc3indel or Samhd1indel, as analyzed by flow cytometry. Of interest, however, Mgaindel mice were detected to have increased germinal center (B220+CD95+CD38-) and marginal zone (B220+CD21highCD23-) splenic B cells, and also showed increased B1a (CD5+ B220low CD23- CD43+) and decreased B1b (CD5- B220low CD23- CD43+) cells in the peritoneum (p<0.05, ANOVA). These results indicate that the likely negative regulatory role that Mga exerts on MYC networks may directly impact germinal center formation and cell fate determination in B cells. The overall abundance of edited B cells in spleen and blood of each group was higher (overall median: 17.0%; 90%CI 6.7-58.8%) than the non-targeting control (8.4%; 90%CI 1.6-14.2%) at 4 months post-transplant (n=8/group, p<0.05, ANOVA), and abundance of edited cells increased in peripheral bleeds at 4 vs. 2 months (n=8/group, p<0.05, Wilcoxon signed rank test). This suggests that presence of individual alterations can alter pro-survival pathways in mature B cells, through mechanisms that may, at least partly, be shared across LOFs. To address this question, we analyzed the transcriptional profiles of edited B cell splenocytes (n=3/group), and compared them to their non-edited counterparts (GFP+mCherry- splenocytes from the same animal), identifying a total of ~3900 differentially expressed genes among the 6 groups (p<0.05, paired Student's t test). Notably, changes in gene expression were highly concordant across 5 of the 6 groups (Spearman r >0.37 for each of the 10 pairs of 5 groups), with the exception of Mgaindel, consistent with its unique phenotype, observed in developmental studies. Gene ontology analyses using Enrichr confirmed commonalities in pathway dysregulations across the 5 similar groups of mice (p<0.05), such as modulation of Notch signaling in Chd2indel, Samhd1indel, and Birc3indel, serine/glycine metabolism in Atmindel, Trp53indel, and Chd2indel, and oxidative phosphorylation in Atmindel and Samhd1indel. Unique to Mgaindel, we saw enrichment of the GOs for transcriptional mis-regulation in cancer and cellular senescence, both relevant for tumorigenesis and B cell development. In conclusion, we demonstrate that common LOFs typical of patients with CLL lead to increased cellular fitness in B-cell restricted mouse models, while dysregulating pro-survival pathways relevant to B cell development, CLL pathogenesis and more broadly to tumorigenesis. We are currently exploring phenotypic similarities and differences through tailored functional assays, while addressing the relative contribution of each alteration to CLL development in multiplexed edited mouse lines. Disclosures Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Temporal Regulation of Notch Signaling and Its Influence on the Differentiation of Ex Vivo Cultured Limbal Epithelial Cells

ABSTRACTPurpose: Notch signaling plays a vital role in the differentiation and proliferation of corneal epithelial cells from limbal stem cells. The temporal regulation of Notch signaling during this differentiation remains unknown. Hence, we investigated the importance of temporal activation/blockage of Notch signaling during corneal differentiation.Methods: Human limbal epithelial cultures were established with and without Notch activators (rec-Human Jagged1 Fc chimera) and pharmacological blockers (LY-411575). The modulation of Notch signaling was done at different time points during cell differentiation, which were collected on Day 14 for further analysis of differentiation, proliferation, maturation and apoptosis using RT-qPCR and immunofluorescence staining.Results: The activation of Notch signaling at Day 8 resulted in the highest number of mature corneal epithelial cells (p = .008) and pro-apoptosis marker BAX (p = .0001) with no increase in the number of corneal progenitors, and proliferation marker Ki67 compared to untreated controls. Cultures grown in the presence of Notch signaling blockers showed a significantly higher number of corneal progenitors (p = .0001) and proliferation marker Ki67 (p = .02) but lower corneal epithelial marker CK3/CK12 (p = .0007) and no difference in the pro-apoptotic marker BAX compared to untreated controls.Conclusion: During the differentiation of limbal epithelial cells to the corneal epithelial cell type, Day 8 seems to be a crucial window to modulate Notch signaling for a customized outcome.

3175 – HEMATOPOIETIC STEM CELL-DEPENDENT NOTCH TRANSCRIPTION IS MEDIATED BY P53 THROUGH THE HISTONE CHAPERONE SUPT16

Hematopoietic stem cells (HSCs) are rare cells that can self-renew and differentiate into all blood cell lineages for life. HSCs have long been the focus of developmental and regenerative studies, yet our understanding of the signaling events regulating their specification remains incomplete. In order to identify novel genes and transcription factors involved in hematopoietic specification, we performed a forward genetic screen to identify zebrafish mutants defective in HSC formation. Through large-scale whole mount in situ hybridization based screens followed by RNA-sequencing-based linkage mapping, we identified that supt16h, a component of the FAcilitates Chromatin Transcription (FACT) complex, is required for HSC formation. Zebrafish supt16h mutants express reduced levels of Notch signaling components, genes essential for HSC development, due to abrogated transcription. Although cellular functions of Supt16h is generally known for regulating transcription and reorganizing nucleosomes to alter chromatin accessibility, global chromatin accessibility in the zebrafish supt16h mutants is unaffected. However, we observe a specific increase in accessibility at the p53 locus leading to an accumulation of P53 protein in the supt16h mutants and abrogation of increased p53 levels in supt16h mutants rescues both loss of Notch and HSC phenotypes. We further demonstrate that P53 levels directly influence expression of the Polycomb Group protein, Phc1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues both loss of Notch and loss of HSC phenotypes in supt16h mutants. Taken together, our results highlight a previously uncharacterized relationship between supt16h, p53, and phc1 to specify HSCs via modulation of Notch signaling.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.