Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination (RNA-Seq of differentiated NSC after lnc-OPC knockdown)
ABSTRACT: To quantitative analysis of transcriptome changes caused by lnc-OPC knockdown during OPC differentiation from NSC, lentivirus-based short hairpin RNAs were used to knockdown the lnc-OPC expression in a neural stem cell culture . Subsequently, puromycin-selected NSCs were differentiated to OPC in culture for three days.RNA-Seq was performed on the polyadenylated fraction of RNA isolated from cell samples. DEseq was used for differential gene expression analysis caused by lnc-OPC knockdown. GO functional term enrichment analysis of differential gene expression caused by lnc-OPC knockdown, revealed significant enrichment of ‘oligodendrocyte development’, ‘oligodendrocyte differentiation’, ‘glia cell development’, and ‘axon ensheathment’ terms that are associated with oligodendrogenesis. mRNA profiles of differentiiated NSC samples after lnc-OPC knockdown by RNA-sequencing.
Project description:Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neuron. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. Overall design: DEG analysis of primary OPC and OL populations, 5 biological replicates per population
Project description:To identify factors involved in OPC senescence, we compared gene expressions between OPC-CG4, OPC-FCS and OPC-Rev. Overall design: We established OPC senescence model system, in which OPC become senescent in the presence of high concentration of FCS. This phenotypes were kept even when the medium was switched to their optimal serum-free medium.
Project description:Like neurons, oligodendrocytes (OL) are cells with elaborate morphology that probably require asymmetrical spatial regulation of biological processes. Formation of membrane protrusions is critical for OL development and interaction with axons. We hypothesized that the enrichment of specific mRNAs in protrusions of oligodendrocyte precursor cells (OPC) is important for morphological differentiation, thus having an impact in myelination. To explore this hypothesis, we established a modified Boyden chamber system to physically separate soma from membrane protrusions of rat primary OPC cultured in vitro for 24h. We performed a whole transcriptome analysis (RNAseq) of primary rat OPC soma and membrane protrusion fractions and found a subcellular enrichment of mRNAs in these structures during initial protrusion formation. At the very initial stage of OPC protrusion extension, there is a significant subcellular enrichment of transcripts encoding proteins related to cellular component assembly and cytoskeleton organization, particularly of actin-related molecules. This suggests that the regulation of the cytoskeleton dynamics may be locally controlled in OPCs and probably relevant for their differentiation program. Overall design: Rat OPCs were plated on a modified Boyden chamber system for 24h consisting of a transwell with 1μm pore size with chemo- and haptotactic gradients to physically separate OPC soma and protrusion fractions for RNA sequencing. RNAseq was done on 2 samples for OPC soma and 2 samples of OPC protrusions. Due to very low RNA yields in OPC protrusions samples (typically 9ng per well), each sample consisted in a pool of several experiments.
Project description:To identify factors involved in OPC senescence, we compared gene expressions between OPC-CG4, OPC-FCS and OPC-Rev. We established OPC senescence model system, in which OPC become senescent in the presence of high concentration of FCS. This phenotypes were kept even when the medium was switched to their optimal serum-free medium.
Project description:PRMT5 is important for gliomas, however its physiological function in oligodendrocyte progenitors (OPCs), remains poorly understood. Here we report PRMT5 to be responsible for symmetric di-methylation of histone H4R3 (H4R3me2s) in OPC. PRMT5 depletion via CRISPR/Cas9 decreased H4R3me2s, altered the OPC transcriptome, and decreased OPC survival. Strikingly, these changes were associated with a marked increase of H4K5ac in OPC, but not in gliomas. Consistently, ChIP-sequencing analysis revealed increased genome-wide distribution of H4K5ac in PRMT5 knockout cells. In vitro acetylation assays demonstrated reciprocal inhibition between symmetric arginine methylation and lysine acetylation. Low H4R3me2s and high H4K5ac levels were also detected in OPC in mice with lineage-specific ablation of Prmt5 (Olig1-Cre;Prmt5fl/fl), resulting in severely impaired developmental myelination. Importantly, pharmacological inhibition of acetyltransferase activity partially rescued the effect of Prmt5 deletion on oligodendrocyte-specific gene transcripts. Collectively, we identify PRMT5 as a critical modulator of histone acetylation and OPC differentiation in early developmental myelination. Overall design: (1) mRNA profiles of OPCs with or without PRMT5 expression were generated by deep sequencing, in tetraplicates, using Illumina HiSeq 2500.
Project description:Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination (RNA-Seq of differentiated NSC after lnc-OPC knockdown)
Project description:The zinc finger protein ZFP24 is critical for CNS myelination. Nonetheless, the mechanism by which ZFP24 controls myelination is unknown. Here we use chromatin IP (ChIP) to map ZFP24 binding sites in oligodendrocyte progenitor cells (OPC) and differentiated oligodendrocytes (OLG). We find that ZFP24 directly binds the enhancer regions of genes important for oligodendrocyte differentiation and myelination and mediates their expression. We demonstrate that ZFP24 undergoes phosphorylation and dephosphorylation in oligodendrocyte lineage cells and that ZFP24 binding to DNA is controlled by its phosphorylated state such that only the non-phosphorylated form of the protein, predominantly found in mature oligodendrocytes, mediates expression of myelin protein genes. We have also identified key ZFP24 downstream target genes. Among these, we show that enforced expression of the crucial myelin transcription factor MYRF can rescue myelin proteins gene expression in ZFP24 -ablated cells. Our data also suggest that ZFP24 display overlapping genomic binding sites with the transcription factors MYRF, SOX10, and OLIG2 which are known to control terminal differentiation of oligodendrocytes. Though the human genome contains roughly 700 C2H2-containing zinc finger proteins, the DNA-binding sequences and the biological functions of the vast majority of them are unknown. Our findings provide a direct molecular mechanism by which dephosphorylation of ZFP24 mediates its binding to enhancer regions of genes important for oligodendrocyte differentiation and myelination, controls their expression, and as a result, regulates oligodendrocyte differentiation and CNS myelination. Overall design: Chromatin IP for ZFP24 was performed on mouse Oligodendrocyte progenitor cells (OPC) and mature (differentiated) oligodendrocyte (OLG). Corresponding Zfp24 null cells were used as control, as well as pooled chromatin input.
Project description:microRNAs (miRNAs) have been implicated in oligodendrogenesis and demyelinating diseases; however, identification of individual miRNAs responsible has remained elusive. Through targeted mutagenesis, we find that miR-219 is required for proper oligodendrocyte differentiation and myelination in vivo. Deletion of miR-338 together with miR-219 further exacerbates hypomyelination phenotypes. Temporally specific ablation reveals a critical role for miR-219 in oligodendrocyte remyelination after demyelination, while overexpression of miR-219 promotes precocious oligodendrocyte maturation and myelin regeneration. Accordingly, administration of miR-219 mimics to lysolecithin-induced demyelinating lesions in the murine spinal cord and brain enhances myelin restoration. Through integrating analyses of transcriptome profiling and biotin-affinity miRNA pull-down, we identify a set of stage-specific targets of miR-219, including Etv5 and Lingo1, as oligodendrocyte differentiation inhibitors. Inhibiting Etv5 and Lingo1 leads to a partial rescue of differentiation defects observed in miR-219-mutant OLs in vitro. Together, our findings identify context-specific miRNA-regulated checkpoints that control CNS myelinogenesis and myelin repair. Overall design: 4 RNA-Seq samples from P12 optic nerves of Ctrl and miR-219 dCKO mice (duplicates, Ctrl and dCKO); 4 RNA-Seq samples from rat OPC with Ctrl or miR-219 mimic transduction (duplicates, Ctrl and miR-219 mimic); 4 RNA-Seq samples from rat iOL with Ctrl or miR-219 mimic transduction (duplicates, Ctrl and miR-219 mimic); and 2 RNA-Seq samples of biotin-miR-219 pull down from rat OPC (ctrl and miR-219 pull down).