Transcription profiling of mouse secondary neurospheres cultured from cells taken from mouse forebrains was compared between FoxO3+/+ (wildtype) and FoxO3-/- (null mutant) mice from the FVB/N background
ABSTRACT: In the nervous system, neural stem cells (NSC) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3-/- mice have fewer NSC in vivo than wild type counterparts. NSC isolated from adult FoxO3-/- mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSC suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSC might have important implications for counteracting brain aging in long-lived species. Experiment Overall Design: mRNA expression from secondary neurospheres cultured from cells taken from mouse forebrains was compared between FoxO3+/+ (wildtype) and FoxO3-/- (null mutant) mice from the FVB/N background.
Project description:In the nervous system, neural stem cells (NSC) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3-/- mice have fewer NSC in vivo than wild type counterparts. NSC isolated from adult FoxO3-/- mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSC suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSC might have important implications for counteracting brain aging in long-lived species. Overall design: mRNA expression from secondary neurospheres cultured from cells taken from mouse forebrains was compared between FoxO3+/+ (wildtype) and FoxO3-/- (null mutant) mice from the FVB/N background.
Project description:Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility to generate patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Considering that ectopic expression of c-Myc causes tumourigenicity in offspring and retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here, we show that adult mouse neural stem cells (NSCs) express higher endogenous levels of Sox2 and c-Myc than ES cells and that exogenous Oct4 together with either Klf4 or c-Myc are sufficient to generate iPS cells from NSCs. These two-factor (2F) iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germline, and form chimeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors. Experiment Overall Design: 8 hybridizations in total. Experiment Overall Design: NSC derived iPS cells by 2 factors (Oct4 and Klf4) in triplicate: Experiment Overall Design: - iPS cell_2F_1 Experiment Overall Design: - iPS cell_2F_2 Experiment Overall Design: - iPS cell_2F_3 Experiment Overall Design: Embryonic Stem cells (ESC) in triplicate: Experiment Overall Design: - ESC_1 Experiment Overall Design: - ESC_2 Experiment Overall Design: - ESC_3 Experiment Overall Design: NSC cultures in duplicates: Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: NSC derived iPS cells by 4 factors (Oct4, Sox2, c-Myc and Klf4) in triplicate: Experiment Overall Design: - iPS cell_4F_1 Experiment Overall Design: - iPS cell_4F_2 Experiment Overall Design: - iPS cell_4F_3
Project description:We showed that the transcription factor Foxo3 played a specific role in the polarization of CD4+ T cells towards pathogenic Th1 cells producing both interferon-γ (IFN-γ) and granulocyte monocyte colony stimulating factor (GM-CSF). To understand the molecular mechanisms whereby Foxo3 controls CD4+ T cell differentiation, unbiased analysis of genes differentially expressed in Foxo3-deficient vs. Foxo3-sufficient CD4+ T cells was achieved using both resting and activated CD4+ T cells obtained following 12 or 24 hours of stimulation with anti-CD3 mAbs. Overall design: Gene expression analysis were performed on purified naive CD4+ T cells from Foxo3-/- (n=4) or WT (n=4) littremate controls either unstimulated (T0) or stimulated with 2 μg/ml of anti-CD3 mAbs for 12 (T12) or 24 (T24) hours
Project description:FOXO transcription factors are central regulators of longevity from worms to humans. FOXO3 – the FOXO isoform associated with exceptional human longevity – preserves adult neural stem cell pools. Here we identify FOXO3 direct targets genome-wide in primary cultures of adult neural progenitor cells (NPCs). Interestingly, FOXO3-bound sites are enriched for motifs for bHLH transcription factors and FOXO3 shares common targets with the pro-neuronal bHLH transcription factor ASCL1/MASH1 in NPCs. Analysis of the chromatin landscape reveals that FOXO3 and ASCL1 are particularly enriched at the enhancers of genes involved in neurogenic pathways. Intriguingly, FOXO3 inhibits ASCL1-dependent neurogenesis in NPCs and direct neuronal conversion in fibroblasts. FOXO3 also restrains neurogenesis in vivo. Our study identifies a genome-wide interaction between the pro-longevity transcription factor FOXO3 and the cell fate determinant ASCL1, and raises the possibility that FOXO3’s ability to restrain ASCL1-dependent neurogenesis may help preserve the neural stem cell pool. ChIP-seq profiles of two transcription factors (FOXO3 and ASCL1) and three histone marks (H3K4me1, H3K4me3 and H3K27me3) in adult mouse neural progenitor cells.
Project description:The iNSC cells are two clones generated from the same MEF line. Therefore, we conducted one analysis that compared the two clonal lines and a separate analysis that compared iNSC vs. NSC, iNSC vs. MEF, and NSC vs. MEF. Both were single factor ANOVAs, the first compared two groups (the iNSC lines) and the second had three groups. For the second analysis, we then used linear contrasts to extract the information about differences between all pairs (e.g. iNSC vs. NSC). Looking at the iNSC lines, the correlations between samples from different clonal lines are as high as the correlations between samples from within a clonal line. Given this, we think that the analysis that combines all 6 of them to compare against the other cell types is appropriate. Array Platform: Affymetrix Mouse Gene 1.0 ST Samples: A total of 12 arrays array# filename genotype 1 01.iNSC1.1.CEL iNSC 2 02.iNSC1.2.CEL iNSC 3 03.iNSC1.3.CEL iNSC 4 04.iNSC2.1.CEL iNSC 5 05.iNSC2.2.CEL iNSC 6 06.iNSC2.3.CEL iNSC 7 07.WT.NSC.1.CEL NSC 8 08.WT.NSC.2.CEL NSC 9 09.WT.NSC.3.CEL NSC 10 10.WT.MEFs.1.CEL MEF 11 11.WT.MEFs.3.CEL MEF 12 12.WT.MEFs.5.CEL MEF
Project description:We generated 2 Gb of high-quality sequencing data (~1 Gb per sample) and catalogued the expression profiles of 48,162 annotated human genes in each sample. The analysis showed differences of transcriptomes between Control and Geminin/FoxO3 co-depletion expression changes. We identified numerous differentially expressed genes that exhibited distinct expression patterns. These genes are appealing candidates for further investigation of the gene expression and associated regulatory mechanisms by Geminin and FoxO3 . Overall design: MDA-MB-231 mRNA profiles of Control, FoxO3 and Geminin/FoxO3 co-depletion were generated by deep sequencing, using Illumina HiSeq4000.
Project description:We have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. We detected significantly increased transcription of a subset of interferon-stimulated genes (ISGs) under basal conditions in Foxo3-null macrophages when compared to their wild type (WT) counterparts, suggesting that FOXO3 functions as a repressor of these genes. Stimulation of Foxo3-null macrophages with poly-IC (PIC) further increased the levels of this subset of ISGs, and also revealed the transcription of additional ISGs. C57BL/6 mice were obtained from Jackson Laboratories. Foxo3-/- mice in the FVB background were obtained from MMRRC and were backcrossed to C57BL/6 mice at least 5 times to generate congenic mice. C57BL/6 Foxo3+/- heterozygotes were intercrossed to generate Foxo3-/- mice. Mice were maintained at the animal facility of the Institute for Systems Biology and used at 8-12 weeks of age. All animals were housed and handled according to the approved protocols of the University of Washington and Institute for Systems Biology's Institutional Animal Care and Use Committees. Bone marrow macrophages from wildtype and Foxo3 knock-out mice were stimulated with PIC or left untreated. 3 replicates per group.
Project description:FOXO transcription factors are key players in diverse cellular responses affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into mechanisms of FOXO regulated gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II (RNAPII) changes shows FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNAPII ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to and activates enhancers as shown by the presence of and changes in enhancer-specific histone modifications and RNAPII occupancy. In addition, FOXO3-mediated enhancer regulation correlates with regulation of adjacent genes and existence of chromatin loops between FOXO3 bound enhancers and regulated genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. seven 4C view point were analyzed on DLD1 colon carcinoma cells containing 4OH-Tamoxifen inducible FOXO3A3-ER (DL23 cells, Kops et al., 2002, Mol Cell Biol), to investigate 3D topology around FOXO3 bound regions and FOXO3 regulated genes before and 4 hours after addition of tamoxifen. 4C procedure, as published before (Splinter et al., 2001, Genes Dev). Cells are cross linked using 1% formaldehyde for 10min at room temperature, nuclei are isolated, after which chromatin is digested with DpnII and subsequently ligated under diluted conditions. After reversal of the cross links the DNA is purified and treated with the second restriction enzyme treatment (Csp). After a second re-ligation step the sample is purified and ligated fragments are analyzed by inverse PCR.
Project description:The ensemble of Foxo3-regulated genes in the erythroid G1E-ER-GATA-1 cell line was determined by knocking down Foxo3 using siRNA, and measuring genome wide transcription by microarray analysis G1E-ER-GATA-1 cells were treated with control or Foxo3-specific siRNA by nucleofection at t = 0 h and t = 24 h. At t = 24 h, cells were treated with ß-estradiol to activate ER-GATA-1. RNA was harvested at t = 48 h and processed for microarray analysis.
Project description:We have previously shown that conditional overexpression of Bmi1 in NSC increases their proliferation both in the developing neocortexand in the postnatal brain (Yadirgi et al. 2011). However, during embryonic development, increased and ectopic proliferation induced by overexpression of Bmi1 in progenitors committed toward a neuronal lineage triggered apoptosis, leading eventually to a reduced overall brain size (Yadirgi et al. 2011). These findings –increased proliferation of neural stem/progenitor cells (NSPC) and apoptosis of neuronal committed progenitors - could be faithfully reproduced in an in vitro assay where NSPC isolated from Nestin-Cre; STOP FloxBmi1 embryos are cultured in floating conditions in the presence of EGF and FGF2 (neurosphere assay). We isolated NSPC from Nestin-Cre;STOP FloxBmi1 E16.5 neocortices and cultured them briefly under neurosphere inducing conditions. We then analysed their transcriptome by whole-genome Illumina platform mouse v2 and compared it to the transcriptome of NSPC isolated from non-transgenic or single transgenic littermates. The objective of this analysis was to identify genes differentially expressed upon overexpression of the PcG gene Bmi1 in neural stem/progenitor cells. 4 samples, 2 condition with 2 biological replicates per condition