Project description:We utilized RNA-seq to identify differential gene expression in Vγ9 Vδ2 gdT cells isolated from human PBMCs upon TCR stimulation. We find that ~30% genes are commonly up- and down-regulated between different phosphoantigen treatments (p<0.05). Inhibition of Notch signaling alongside TCR activation results in downregulation of many effector genes in Vγ9 Vδ2 T cells.
Project description:This randomized phase I/II clinical trial is studying the side effects and best dose of gamma-secretase/notch signalling pathway inhibitor RO4929097 when given together with vismodegib and to see how well they work in treating patients with advanced or metastatic sarcoma. Vismodegib may slow the growth of tumor cells. Gamma-secretase/notch signalling pathway inhibitor RO4929097 may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. Giving vismodegib together with gamma-secretase/notch signalling pathway inhibitor RO4929097 may be an effective treatment for sarcoma.
Project description:ASCL1 mediates neuronal differentiation of GBM stem cell (GSC) cultures upon Notch signalling inhibition. We sought to identify gene expression changes that were specific to ASCL1 function. In this dataset, we include RNA-seq data obtained from GSC cultures harbouring wildtype or CRISPR-deletion of ASCL1. We assessed differential gene expression between wildtype and ASCL1-knockout after treatment with gamma-secretase inhibitor for 7 days.
Project description:Gamma-delta (gd) T cells from pooled mouse lymph nodes and spleens were isolated and FACS-sorted for Vg1+Ly6C-, Vg1+Ly6C+, Vg4+Ly6C- and Vg4+Ly6C+ subsets of bulk CD27+ gdT cells.
Project description:The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, its role during hypothalamus formation along with its downstream effectors remains poorly defined. Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. This data gives essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network. Four sets of samples were used for analysing transcriptome changes in DAPT treated chicl embryos. The rostral part of the head (including the anterior forebrain, optic vesicles and overlying tissue) was collected from control embryos and DAPT-treated embryos at stage HH13.
Project description:The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, its role during hypothalamus formation along with its downstream effectors remains poorly defined. Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. This data gives essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network.
Project description:Notch signalling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighbouring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and together with the transcription factor moiety of Notch (NICD) it regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole genome binding profiles were generated for RBPJ, its coactivator p300, NICD and the histone H3 modifications H3K4me3, H3K4me1 and H3K27ac in myogenic cells under active or inhibitory Notch signalling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300, and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signalling mediate their activities and consequently impact on cell fate decisions. ChIP (chromatin immunoprecipitation) is followed by deep sequencing to generate genome-wide patterns of RBP-J binding in mouse C2C12 cells under various conditions. Cells were either Notch activated by exposure to immobilized ligand or by overexpression of NICDGFP, or Notch inhibited by treatment with DAPT. Notch activation and inhibition treatments were applied for 6h and 24h. In addition to RBP-J, p300 and NICDGFP were profiled by ChIP-Seq and gene expression was assessed by RNA-Seq.
Project description:Notch signalling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighbouring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and together with the transcription factor moiety of Notch (NICD) it regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole genome binding profiles were generated for RBPJ, its coactivator p300, NICD and the histone H3 modifications H3K4me3, H3K4me1 and H3K27ac in myogenic cells under active or inhibitory Notch signalling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300, and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signalling mediate their activities and consequently impact on cell fate decisions.
Project description:In Hydra, Notch inhibition causes defects in head patterning and prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. To understand the molecular mechanisms by which the Notch pathway regulates these processes, we performed RNA-seq and identified genes that are differentially regulated in response to 48 h of treating the animals with the Notch inhibitor DAPT. To identify candidate direct regulators of Notch signalling, we profiled gene expression changes that occur during subsequent restoration of Notch activity and performed promoter analyses to identify RBPJ transcription factor-binding sites in the regulatory regions of Notch-responsive genes. Interrogating the available single-cell sequencing data set revealed the gene expression patterns of Notch-regulated Hydra genes. Through these analyses, a comprehensive picture of the molecular pathways regulated by Notch signalling in head patterning and in interstitial cell differentiation in Hydra emerged. As prime candidates for direct Notch target genes, in addition to Hydra (Hy)Hes, we suggest Sp5 and HyAlx. They rapidly recovered their expression levels after DAPT removal and possess Notch-responsive RBPJ transcription factorbinding sites in their regulatory regions