Project description:Polycomb repressive complexes (PRCs) are important chromatin regulators of ES cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B, and has been suggested to participate in localizing Polycomb complexes to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Here we have used ES cells conditionally deficient in RYBP to investigate RYBP function. Chromosome immunoprecipitation on a chip (ChIP-chip) of RYBP and microarray experiments were performed using wild type and knocked-out ES cells. Gene expression profiling of WT, conditionally deficient in RYBP with or without Yaf2 RNAi, and ChIP-chip of RYBP on promoters of WT, Dnmt1-KO or Eed-KO ES cells.
Project description:We present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Multiple ESC and EGC cell lines were cultured without feeders on gelatin coated, 6-well plates, 100,000 cells/well (10,000 cells/cm2) in 3 conditions: 1) in complete ES medium: DMEM, 15% FBS; LIF (ESGRO) 1000 u/ml; 1mM sodium pyruvate; 0.1 mM NEAA, 2 mM glutamate, 0.1 mM beta-mercapto ethanol, and penicillin/streptomycin (50U/50ug per ml); 2) in ES medium without LIF; 3) in complete ES medium plus 1 uM RA. Cells were incubated at 37 0C; 5% CO2. Medium was changed daily. On the day 3 Trizol was added, RNA extracted using Phase lock gelTM (Eppendorf) columns according to manufacturer protocol. RNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in DEPC dH2O.
Project description:Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein Geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized both neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, Geminin antagonized the ability of neural bHLH transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, Geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. Geminin is highly expressed in undifferentiated neuronal precursor cells but is downregulated prior to differentiation. Therefore, these data support a model whereby Geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis. A mouse embryonic stem (ES) cell line for inducible knockdown of the small nucleoprotein Geminin was utilized. ES cells were used to generate neural precursor cells by monolayer culture in N2B27 media for 5 days, and doxycycline-inducible knockdown of Geminin was performed from day 3. Changes in gene expression resulting from Geminin knockdown were assessed by RNA sequencing. Three experimental replicates were generated for Geminin knockdown (plus Dox) with a corresponding no-Dox control. These were subjected to sequencing, and data were analyzed using TopHat and Cufflinks/Cuffdiff. Transcripts were considered as differentially expressed upon Gem knockdown if data met statistical significance cutoffs in Cuffdiff (sufficient sequence alignments were obtained for analysis and transcript had significant change in FPKM value (normalized transcript abundance; fragments per kb of exon per million fragments mapped) between the no Dox and plus Dox sample pairs) in at least two of the three replicates.
Project description:This SuperSeries is composed of the following subset Series: GSE14559: Timed induction of 50 transcription factors in ES cells reveals a common mechanism to initiate differentiations GSE14586: Cdx2 Binding Sites On Cdx2 Expressing ES Cells GSE16148: Timed induction of 10 transcription factors - ES time series data Refer to individual Series
Project description:Cell type-specific master transcription factors (MTFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that all three subunits of the ubiquitous heterotrimeric CCAAT-binding NF-Y complex are required for the maintenance of embryonic stem cell (ESC) identity, and establish NF-Y as a novel component of the core pluripotency network. Genome-wide occupancy and transcriptomic analyses in ESCs and neurons reveal that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with MTFs. Mechanistically, NF-Y's distinctive DNA-binding mode promotes MTF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a novel function for NF-Y in promoting chromatin accessibility, and suggest that other proteins with analogous structural and DNA-binding properties may function in similar ways. Genome-wide mapping of NF-YA, NF-YB, and NF-YC subunits of the NF-Y complex in mouse ESCs, and microarray gene expression profiling of control knockdown (KD), NF-YA KD, NF-YB KD, NF-YC KD, and NF-YA/NF-YB/NF-YC triple KD ESCs.
Project description:Parkinson’s disease (PD) is characterized by a selective loss of dopamine (DA) neurons in the human midbrain causing motor dysfunctions. The exact mechanism behind dopaminergic cell death is still not completely understood and, so far, no cure or neuroprotective treatment for PD is available. Recent studies have brought attention to the wide array of bioactive molecules produced by mesenchymal stem cells (MSCs), generally referred to as the secretome. Herein, we evaluated whether human MSCs-bone marrow derived (hBMSCs) secretome would be beneficial in a PD pre-clinical model, when compared directly with cell transplantation of hBMSCs alone. We used a 6-hydroxydpomanie (6-OHDA) rat PD model, and motor behavior was evaluated at different time points after treatments (1, 4 and 7 weeks). The impact of the treatments in the recovery of DA neurons was estimated by determining TH-positive neuronal densities in the substantia nigra and fibers in the striatum, respectively, at the end of the behavioral characterization. Furthermore, we determined the effect of the hBMSCs secretome on the neuronal survival of human neural progenitors in vitro, and characterized the secretome through proteomic-based approaches. This work demonstrates that the injection of hBMSCs secretome potentiated the histological recovery of DA neurons, when compared to transplantation of hBMSCs themselves, a fact that probably explains the improved behavioral performance of secretome-injected animals in the staircase test. Moreover, we observed that hBMSCs secretome induces higher levels of in vitro neuronal differentiation. Finally, the proteomic analysis revealed that hBMSCs secrete a variety of important exosome-related molecules such as those related with the ubiquitin-proteasome and histone systems. Overall, this work provided important insights on the potential use of hBMSCs secretome as a therapeutic tool for PD, and further confirms the importance of the secreted molecules rather than the transplantation of hBMSCs for the observed positive effects. These could be likely through normalization of defective processes in PD, namely proteostasis or altered gene transcription, which lately can lead to neuroprotective effects.
Project description:Stem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks. To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs following the initiation of commitment and integrated these data with known genome-wide transcription factor binding. Effects of forced under- or over-expression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells. By using this integrative approach we have, for the first time, formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks. Experiment Overall Design: 1, 3, and 5 days LIF differentiated ESCs, and 1 and 2 days RA differentiated ESCs
Project description:Rybp binding in wild-type, Eed-KO, and Dnmt1-KO ES cells ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent-014716 and Agilent-014717, Palo Alto, Calif., USA). ESCs were subjected to ChIP assay using a Rybp antibody (Santa Cruz; H-115). Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification as described previously (van Bakel et al., 2008). Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions.