Neuronal Differentiation Of P19 Cells Research Articles

Esco2 promotes neuronal differentiation by repressing Notch signaling

Esco2 is an acetyltransferase that is required for the establishment of sister chromatid cohesion. Roberts–SC phocomelia (RBS) syndrome caused by the mutations of Esco2 gene, is an autosomal recessive development disorder characterized by growth retardation, limb reduction and craniofacial abnormalities including cleft lip and palate. Here, we show that Esco2 protein co-immunoprecipitates with Notch but not with CBF1. Esco2 represses the transactivational activity of Notch protein in an acetyltransferase-independent manner. Chromatin immunoprecipitation experiments suggest that Esco2 might regulate the activity of NICD–CBF1 via attenuating NICD binding to CBF1 on the promoter of Hes1, the downstream target gene of Notch. Furthermore, we demonstrate that the overexpression of Esco2 promotes the neuronal differentiation of P19 embryonic carcinoma cells and C17.2 neural progenitor cells and the knockdown of Esco2 by siRNA blocks the differentiation. The inhibitory effects of Notch protein on neuronal differentiation of P19 cells was suppressed by Esco2 overexpression. Taken together, our study suggests that Esco2 may play an important role in neurogenesis by attenuating Notch signaling to promote neuronal differentiation.

Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cyclic AMP-dependent protein kinase

cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβ protein. PKIβ played a very specific role in the Ascl1-mediated differentiation process as other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells.

A novel RING finger protein, Znf179, modulates cell cycle exit and neuronal differentiation of P19 embryonal carcinoma cells

Znf179 is a member of the RING finger protein family. During embryogenesis, Znf179 is expressed in a restricted manner in the brain, suggesting a potential role in nervous system development. In this report, we show that the expression of Znf179 is upregulated during P19 cell neuronal differentiation. Inhibition of Znf179 expression by RNA interference significantly attenuated neuronal differentiation of P19 cells and a primary culture of cerebellar granule cells. Using a microarray approach and subsequent functional annotation analysis, we identified differentially expressed genes in Znf179-knockdown cells and found that several genes are involved in development, cellular growth, and cell cycle control. Flow cytometric analyses revealed that the population of G0/G1 cells decreased in Znf179-knockdown cells. In agreement with the flow cytometric data, the number of BrdU-incorporated cells significantly increased in Znf179-knockdown cells. Moreover, in Znf179-knockdown cells, p35, a neuronal-specific Cdk5 activator that is known to activate Cdk5 and may affect the cell cycle, and p27, a cell cycle inhibitor, also decreased. Collectively, these results show that induction of the Znf179 gene may be associated with p35 expression and p27 protein accumulation, which lead to cell cycle arrest in the G0/G1 phase, and is critical for neuronal differentiation of P19 cells.

Myt/NZF family transcription factors regulate neuronal differentiation of P19 cells

During mammalian central nervous system development, neural stem cells differentiate and then mature into various types of neurons. Myelin transcription factor (Myt)/neural zinc finger (NZF) family proteins were first identified as myelin proteolipid protein promoter binding factors and were shown to be involved in oligodendrocyte development. In this study, we found that Myt/NZF family molecules were expressed during neuronal differentiation in vivo and in vitro. Transient over-expression of Myt/NZF family genes could convert undifferentiated P19 cells into neurons without induction by retinoic acid (RA), and the ability of these genes to induce neuronal differentiation was comparable to that of Neurog1 and Neurod1. Additionally, we found that St18 (or NZF-3) was induced by several bHLH transcription factors. When NZF-3 and Neurog1 were co-expressed in P19 cells, the rate of neuronal differentiation was significantly increased. These data suggest not only that NZF-3 works downstream of Neurog1 but also that it plays a crucial role together with Neurog1 in neuronal differentiation.

Comprehensive Analysis of Alternative Splicing and Functionality in Neuronal Differentiation of P19 Cells

BackgroundAlternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system.Methodology/Principal FindingsThe purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed.Conclusions/SignificanceAlternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

SR Proteins Induce Alternative Exon Skipping through Their Activities on the Flanking Constitutive Exons

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.

Stat3 is essential for neuronal differentiation through direct transcriptional regulation of the Sox6 gene

The transcription factor Signal Transducer and Activator of Transcription 3 (Stat3) functions in various cellular processes including neuronal differentiation. We show that the SRY-box containing gene 6 (Sox6) gene, important for neuronal differentiation, is a direct target gene of Stat3. We demonstrate that in response to ligand stimulation, Stat3 binds to the Sox6 promoter and induces its expression. Furthermore, Stat3 is activated and Sox6 is induced during neuronal differentiation of P19 cells in the absence of exogenous ligand treatment. Moreover, using an RNA interference approach, we show that Stat3 is required for Sox6 expression during neuronal differentiation.

Jmjd3 activates Mash1 gene in RA‐induced neuronal differentiation of P19 cells

Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri-methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix-loop-helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA-treated P19 cells. Over-expression of Jmjd3 in P19 cells also significantly enhances the RA-induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA-induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co-Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA-induced neuronal differentiation of P19 cells.

Mammalian Numb-interacting Protein 1/Dual Oxidase Maturation Factor 1 Directs Neuronal Fate in Stem Cells

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

Role of Acetylated p53 in Regulating the Expression of map2 in Retinoic Acid-induced P19 Cells

To investigate the regulatory mechanisms of acetylated p53 in the expression of microtubule-associated protein-2 (MAP2) in neuronal differentiation of P19 cells induced by all-trans retinoic acid (RA). Neuronal differentiation of P19 cells was initiated with 4-day RA treatment. Immunofluorescence, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, and map2 promoter driven luciferase assay were performed to detect the expression and relative promoter activity of MAP2 in those RA-treated cells. Real-time PCR-based chromatin immunoprecipitation assay (ChIP) was carried out to reveal the specific recruitment of acetylated p53 onto its binding sites on map2 promoter. The expression of MAP2 was markedly increased in RA-induced P19 cells. The map2 mRNA increased 34-fold after 4 days of RA treatment and 730-fold 2 days after the treatment, compared with the cells without RA treatment (control). p53 was recruited to the promoter of map2 gene in acetylated form and thereby enhanced its promoter activity. p300/CBP associated factor (PCAF) was found induced in RA-treated cells and enriched in the nucleus, which might contribute to the acetylation of p53 in the regulation of map2 gene. Acetylated p53 may participate in regulating the expression of map2 in RA-induced differentiation of P19 cells. PCAF is possibly involved in this process by mediating the acetylation of p53.

Mouse microRNA-124 regulates the expression of Hes1 in P19 cells

MicroRNAs (miRNAs) belong to a conserved class of small non-coding RNAs that are typically 18-25 nucleotides long. They are found in both animals and plants. These small RNAs can regulate gene expression at translational level by interacting with their target messenger RNAs, and they play an essential role in the development of plants and animals. To date, more than 200 miRNAs have been identified in mammals; however, their mRNA targets have not yet been identified. In this study, we demonstrate that the expression of Hes-1, which is a basic helix-loop-helix transcriptional repressor, is regulated by miRNA-124 in P19 cells. Reduction in the levels of miR-124 mediated by locked nucleic acids resulted in the accumulation of Hes-1 and hindered the retinoic acid-induced neuronal differentiation of P19 cells. Thus, our results indicate that miR-124 regulates the expression of Hes-1 at the post-transcriptional level and is involved in the retinoic acid-induced neuronal differentiation of P19 cells.

Reciprocal roles of SIRT1 and SKIP in the regulation of RAR activity: implication in the retinoic acid-induced neuronal differentiation of P19 cells

Human sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that participates in cell death/survival, senescence and metabolism. Although its substrates are well characterized, no direct regulators have been defined. Here, we show that SIRT1 associates with SKI-interacting protein (SKIP) and modulates its activity as a coactivator of retinoic acid receptor (RAR). Binding assays indicated that SKIP interacts with RAR in a RA-dependent manner, through a region that overlaps the binding site for SIRT1. SKIP augmented the transcriptional activation activity of RAR by cooperating with SRC-1, and SIRT1 suppressed SKIP/SRC-1-enhanced RAR transactivation activity. The suppression was dependent on the deacetylase activity of SIRT1 and was enhanced by a SIRT1 activator, resveratrol. In contrast, the suppression was relieved by SIRT1 knockdown, overexpression of SKIP and treatment with a SIRT1 inhibitor, splitomicin. Upon SKIP overexpression, the recruitment of SIRT1 to the endogenous RARβ2 promoter was severely impaired, and SKIP was recruited to the promoter instead. Finally, resveratrol treatment inhibited RA-induced neuronal differentiation of P19 cells, accompanied by reductions in the neuronal marker nestin and a RAR target gene, RARβ2. This inhibition was relieved by either knockdown of SIRT1 or overexpression of SKIP. These data suggest that SIRT1 and SKIP play reciprocal roles in the regulation of RAR activity, which is implicated in the regulation of RA-induced neuronal differentiation of P19 cells.

Systematic gene regulation involving miRNAs during neuronal differentiation of mouse P19 embryonic carcinoma cell

MicroRNAs (miRNAs) are small noncoding RNA and play an essential role in gene regulation. In this study, we investigated regulation of gene expression during neuronal differentiation of mouse P19 embryonic carcinoma cells and described a systematic pathway of gene regulation involving miRNAs. In the pathway, downregulation of Lin28 involved in blocking the let-7 maturation and upregulation of let-7 occur following induction of the differentiation, thereby triggering suppression of the downstream High Mobility Group A2 ( Hmga2) gene expression via activation of gene silencing mediated by let-7. Our data further suggest that miR-9, as well as miR-125b, participate in the reduction of the Lin28 expression. The gene regulation involving miRNAs likely contributes to a rapid and programmed change in gene expression in neuronal differentiation of P19 cells.

Noninvasive imaging of microRNA124a‐mediated repression of the chromosome 14 ORF 24 gene during neurogenesis

The function of microRNAs (miRNAs) is translational repression or mRNA cleavage of target genes by binding to 3'-UTRs of target mRNA. In this study, we investigated the functions and the target genes of microRNA124a (miR124a), and imaged the miR124a-mediated repression of chromosome 14 open reading frame24 (c14orf24, unknown function) during neurogenesis, using noninvasive luciferase systems. The expression and functions of miR124a were investigated in neuronal differentiation of P19 cells (P19 is a mouse embryonic carcinoma cell line) by qRT-PCR and RT-PCR. The predicted target genes of miR124a were found by searching a bioinformatics database and confirmed by RT-PCR analysis. Remarkable repression of c14orf24 by miR124a was detected during neurogenesis, and was imaged using in vitro and in vivo luciferase systems. The expression of miR124a was highly upregulated during neuronal differentiation. Overexpression of miR124a in P19 cells resulted in a preneuronal gene expression pattern. MicroRNA124a-mediated repression of c14orf24 was successfully monitored during neuronal differentiation. Also, c14orf24 showed molecular biological characteristics as follows: dominant expression in the cytoplasm; a high level of expression in proliferating cells; and gradually decreased expression during neurogenesis. Our noninvasive luciferease system was used for monitoring the functions of miRNAs, to provide imaging information on miRNA-related neurogenesis and the miRNA-regulated molecular network in cellular metabolism and diseases.

ZNF536, a Novel Zinc Finger Protein Specifically Expressed in the Brain, Negatively Regulates Neuron Differentiation by Repressing Retinoic Acid-Induced Gene Transcription

Neuronal differentiation is tightly regulated by a variety of factors. In a search for neuron-specific genes, we identified a highly conserved novel zinc finger protein, ZNF536. We observed that ZNF536 is most abundant in the brain and, in particular, is expressed in the developing central nervous system and dorsal root ganglia and localized in the cerebral cortex, hippocampus, and hypothalamic area. During neuronal differentiation of P19 cells induced by retinoic acid (RA), ZNF536 expression is increased at an early stage, and it is maintained at a constant level in later stages. Overexpression of ZNF536 results in an inhibition of RA-induced neuronal differentiation, while depletion or mutation of the ZNF536 gene results in an enhancement of differentiation. We further demonstrated that ZNF536 inhibits expression of neuron-specific marker genes, possibly through the inhibition of RA response element-mediated transcriptional activity, as overexpression of RA receptor alpha can rescue the inhibitory role of ZNF536 in neuronal differentiation and neuron-specific gene expression. Our studies have identified a novel zinc finger protein that negatively regulates neuron differentiation.

Histone marks and chromatin remodelers on the regulation of neurogenin1 gene in RA induced neuronal differentiation of P19 cells

Neurogenin1 is an important bHLH protein that plays crucial role in neurogenesis. We first show that the expression of ngn1 increases drastically in RA induced neuronal differentiation. During which, a three successive stages of the epigenetic changes surrounding the ngn1 gene are found correlated with a repression to activation of the gene in P19 cells. Recruiting of a repressive histone code H3K27me3 on the ngn1 gene is the dominant change in first repression stage, which is followed by the binding of the active codes of H3K9ac, H3K14ac, and the H3K4me3 in the second and third stages of RA treatment. Additionally, BRM but not BRG1 is specifically recruited to ngn1 gene at the third stage and is positively involved in the RA induced ngn1 expression. We propose that histone modifiers and chromatin remodelers are pivotal in the activation of the ngn1 gene in RA induced differentiation of P19 cells.

MicroRNA 18 and 124a Down-Regulate the Glucocorticoid Receptor: Implications for Glucocorticoid Responsiveness in the Brain

Glucocorticoids (GCs) exert profound effects on a variety of physiological processes, including adaptation to stress, metabolism, immunity, and neuronal development. Cellular responsiveness to GCs depends on numerous factors, including the amount of the glucocorticoid receptor (GR) protein. We tested the hypothesis that micro-RNAs (miRs), a recently discovered group of noncoding RNAs involved in mRNA translation, might control GR activity by reducing GR protein levels in neuronal tissues. We tested a panel of five miRs consisting of 124aa, 328, 524, 22, and 18. We found that miRs 18 and 124a reduced GR-mediated events in addition to decreasing GR protein levels. miR reporter assays revealed binding of miR-124a to the 3' untranslated region of GR. In correspondence, the activation of the GR-responsive gene glucocorticoid-induced leucine zipper was strongly impaired by miR-124a and -18 overexpression. Although miR-18 is expressed widely throughout the body, expression of miR-124a is restricted to the brain. Endogenous miR-124a up-regulation during neuronal differentiation of P19 cells was associated with a decreasing amount of GR protein levels and reduced activity of luciferase reporter constructs bearing GR 3' untranslated regions. Furthermore, we show that miR-124a expression varies over time during the stress hyporesponsive period, a neonatal period when GC signaling is modulated. Our findings demonstrate a potential role for miRs in the regulation of cell type-specific responsiveness to GCs, as may occur during critical periods of neuronal development. Ultimately, our results may provide a better understanding of the etiology of stress-related diseases as well as the efficacy of GC therapy.

Sam68 Regulates a Set of Alternatively Spliced Exons during Neurogenesis

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.

Methyltransferase-inhibition interferes with neuronal differentiation of P19 embryonal carcinoma cells

We have analyzed the importance of substrate methylation by S-adenosylmethionine-dependent methyltransferases for neuronal differentiation of P19 embryonal carcinoma cells. We show that treatment of cells with methyltransferase inhibitor adenosine dialdehyde (AdOx) interferes with neuronal differentiation. Retinoic acid (RA) and AdOx co-treated cells had a decreased number of neurites and a flattened morphology compared with cells differentiated by RA. Also, the amount of neuronal class III tubulin (Tuj1) decreased from 76% to 9.6% with AdOx-treatment. Gene expression levels of wnt-1, brn-2, neuroD, and mash-1 were also down-regulated by AdOx-treatment. But AdOx-treatment did not up-regulate BMP-4 and GFAP genes. Treatment of RA decreased E-cadherin expression during neuronal differentiation. However, in AdOx/RA co-treated cells, E-cadherin expression was restored to the control level. Also, mRNA expression of N-cadherin decreased with AdOx-treatment. Taken together, these data show that methylation reactions might influence the cell-fate decision and neuronal differentiation of P19 cells.

Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells

Generally, the 3'-end of the duplex microRNA (miR) precursor (pre-miR) is known to be stable in vivo and serve as a mature form of miR. However, both the 3'-end (miR9) and 5'-end (miR9*) of a brain-specific miR9 have been shown to function biologically in brain development. In this study, real-time PCR analysis and in vitro/in vivo bioluminescent imaging demonstrated that the upstream region of a primary miR9-1 (pri-miR9-1) can be used to monitor the highly expressed pattern of endogenous pri-miR9-1 during neurogenesis, and that the Luciferase reporter gene can image the unequal expression patterns of miR9 and miR9* seen during the neuronal differentiation of P19 cells. This demonstrates that our bioimaging system can be used to study the participation of miRs in the regulation of neuronal differentiation.