ABSTRACT: Genome-wide sequencing of REST binding (ChIP-seq) in adult hippocampal neural stem cells in vitro a.k.a. HCN cells in quiescent (iQNP) and proliferative (TAP) conditions
Project description:We report the high-throughput transcriptome profiling of induced quiescent (iQNP) and proliferative (TAP) conditions in mammalian adult hippocampal stem cells in culture (HCN cells). By obtaining 145 to 78 million pair-end reads of sequence from isolated RNA from HCN cells in iQNP and TAP conditions. Thereafter, we intersected this gene expression data with REST bound ChIP-seq peaks within +/-10kb of transcription start site of genes also from HCN cells in iQNP and TAP conditions. We find that with +/-10kb of transcription start site of genes, REST efficiently binds neuronal genes and represses them in HCN cells in both iQNP and TAP conditions. Moreover, only in the iQNP REST also binds non-neuronal genes like DNA replication genes. We also report the high-throughput transcriptome profiling of iQNP and TAP condition HCN cells electroporated with a control empty vector or REST knock-down shRNA vector. By obtaining 145 to 78 million pair-end reads of sequence from isolated RNA from HCN cells in electroporated control empty vector or REST knock-down vector in iQNP and TAP conditions. In REST knockdown in iQNP and TAP conditions we found that predominantly non-neuronal genes and neuronal genes were derepressed. This study reveals that in addition to its well known function as a neuronal repressor in non-neuronal tissue, REST can also play other diverse roles in non-neuronal tissues.
Project description:We report the high-throughput profiling of REST binding in mammalian adult hippocampal stem cells in culture (HCN cells). By obtaining 33 to 38 million single-end reads of sequence from chromatin immunoprecipitated DNA, we generated genome-wide REST binding state of HCN cells in induced quiescent (iQNP) and proliferating (TAP) conditions. Thereafter, we annotated the peaks to genomic regions and associated genes. We find that with +/-10kb of transcription start site of genes, REST efficiently binds neuronal genes and represses them in HCN cells in both iQNP and TAP conditions. Moreover, only in the iQNP REST also binds non-neuronal genes like DNA replication genes. This study reveals that in addition to its well known function as a neuronal repressor in non-neuronal tissue, REST can also play other diverse roles in non-neuronal tissues.
Project description:Genome wide transcriptome analysis (RNA-seq) of adult hippocampal neural stem cells in vitro a.k.a. HCN cells in quiescent and proliferative conditions in non-electroporated or electroporated with control empty vector or REST knock-down shRNA vector
Project description:Adult hippocampal neurogenesis is important for certain forms of cognition and failing neurogenesis has been implicated in neuropsychiatric diseases. The neurogenic capacity of hippocampal neural stem/progenitor cells (NSPCs) depends on a balance between quiescent and proliferative states. However, how this balance is regulated remains poorly understood. Here we show that the rate of fatty acid oxidation (FAO) defines quiescence vs. proliferation in NSPCs. Quiescent NSPCs show high levels of carnitine palmitoyltransferase 1a (Cpt1a)-dependent FAO, which is downregulated in proliferating NSPCs. Pharmacological inhibition and conditional deletion of Cpt1a in vitro and in vivo leads to altered NSPC behavior, showing that Cpt1a-dependent FAO is required for stem cell maintenance and proper neurogenesis. Strikingly, experimental manipulation of malonyl-CoA, the metabolite that regulates levels of FAO, is sufficient to induce exit from quiescence and to enhance NSPC proliferation. Thus, the data presented here identify a shift in FAO metabolism that governs NSPC behavior and suggest an instructive role for fatty acid metabolism in regulating NSPC activity.
Project description:Gene expression of mouse hepatic stellate cells was characterized under the following conditions: Quiescent (isolated from normal mouse liver) and reverted (isolated from mouse liver treated with 4 injections of carbontetrachloride followed by 45 day rest period) Affymetrix Mouse 1.0ST gene expression measurements were used to characterize the transcriptomic basis in quiescent hepatic stellate cells, isolated from normal liver, and reverted hepatic stellate cells, isolated from liver treated with 4 injections of CCl4 followed by a 45 day rest period. Gene expression of mouse hepatic stellate cells was characterized under the following conditions: A. Quiescent control hepatic stellate cells (n=4). B. Reverted hepatic stellate cells (n=4).
Project description:Pseudomonas aeruginosa produces the toxic secondary metabolite hydrogen cyanide (HCN) at high cell population densities and low aeration. We have used Affimetrix microarrays to investigate the impact of HCN as a signal in cell-cell communication by comparing the transcriptome of the wild-type strain PAO1 to that of an HCN-negative mutant (PAO6344) under cyanogenic conditions. Total RNA was extracted from cultures of the wild-type strain PAO1 as well as from the isogenic HCN negative mutant (PAO6344), when HCN production was maximal (i.e. end of exponential growth phase). Each RNA pool derived from 3 technical replicates. 1 biological replicate per strain was performed on a different day.
Project description:We have characterized the transcriptome of adult rat hippocampal neural stem and progenitor cell (NSPC) cultures. The NSPCs cultures can be reversibly arrested by the quiescence-promoting signal BMP4. We provide expression data from NSPCs grown as neurospheres for 4 days in vitro in the presence of FGF2 (proliferating NSPCs) or FGF2+BMP4 (quiescent NSPCs) showing that entry into quiescence involves major changes in the transcriptional profile of the cells.
Project description:Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic suite, to statistically quantify singe-cell gene expression along de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of qNSCs, characterized by high niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying adult qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes. Single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny.
Project description:Gene expression of mouse hepatic stellate cells was characterized under the following conditions: Quiescent (isolated from normal mouse liver) and reverted (isolated from mouse liver treated with 4 injections of carbontetrachloride followed by 45 day rest period) Affymetrix Mouse 1.0ST gene expression measurements were used to characterize the transcriptomic basis in quiescent hepatic stellate cells, isolated from normal liver, and reverted hepatic stellate cells, isolated from liver treated with 4 injections of CCl4 followed by a 45 day rest period.
