ABSTRACT
As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor, Ppp1r14a, was dependent on distinct, canonical E-box sequences within the Ppp1r14a promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Neurogenesis , Promoter Regions, Genetic , Animals , Cell Line, Tumor , Embryonal Carcinoma Stem Cells/cytology , Embryonal Carcinoma Stem Cells/metabolism , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins , Mice , Muscle Proteins/genetics , Muscle Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Octamer Transcription Factor-3/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Transcription, GeneticABSTRACT
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 ß-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)ß. PKIß-targeted shRNA constructs both reduced the levels of PKIß 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 PKIß protein, and this rescue required the PKA-specific inhibitory sequence of the PKIß protein. PKIß 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 PKIß. Our results define a novel requirement for PKIß and its inhibition of PKA during neuronal differentiation of P19 cells.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Neoplastic/physiology , Neurons/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , CREB-Binding Protein/metabolism , Carcinoma/pathology , Cell Differentiation/drug effects , Cell Line, Transformed , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/genetics , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Mice , Neurons/drug effects , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Thionucleotides/pharmacology , Transfection , Tubulin/metabolismABSTRACT
The basic helix-loop-helix transcription factor Ascl1 plays a critical role in the intrinsic genetic program responsible for neuronal differentiation. Here, we describe a novel model system of P19 embryonic carcinoma cells with doxycycline-inducible expression of Ascl1. Microarray hybridization and real-time PCR showed that these cells demonstrated increased expression of many neuronal proteins in a time- and concentration-dependent manner. Interestingly, the gene encoding the cell cycle regulator Gadd45gamma was increased earliest and to the greatest extent following Ascl1 induction. Here, we provide the first evidence identifying Gadd45gamma as a direct transcriptional target of Ascl1. Transactivation and chromatin immunoprecipitation assays identified two E-box consensus sites within the Gadd45gamma promoter necessary for Ascl1 regulation, and demonstrated that Ascl1 is bound to this region within the Gadd45gamma promoter. Furthermore, we found that overexpression of Gadd45gamma itself is sufficient to initiate some aspects of neuronal differentiation independent of Ascl1.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/physiology , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins/metabolism , Neurons/physiology , Transcription, Genetic , Animals , Anti-Bacterial Agents/pharmacology , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/drug effects , Doxycycline/pharmacology , Gene Expression Regulation/drug effects , Helix-Loop-Helix Motifs , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Microarray Analysis , Neurons/cytology , Neurons/drug effects , Patch-Clamp Techniques , Promoter Regions, Genetic , Tumor Cells, Cultured , GADD45 ProteinsABSTRACT
Dentate granule cell (DGC) neurogenesis persists throughout life in the mammalian hippocampal dentate gyrus and increases after epileptogenic insults. The DGC layer in human and experimental mesial temporal lobe epilepsy (mTLE) often shows abnormal dispersion and the appearance of hilar-ectopic DGCs. In the pilocarpine mTLE model, hilar-ectopic DGCs arise as a result of an aberrant chain migration of neural progenitors. Reelin is a secreted migration guidance cue that persists in the adult rodent and human hippocampus. We tested the hypothesis that loss of Reelin in the epileptic dentate gyrus leads to aberrant chain migration of DGC precursors. We found that interneuron subsets typically lost in human and experimental mTLE express Reelin, and DGC progenitors express the downstream Reelin signaling molecule Disabled 1 (Dab1). Prolonged seizures decreased Reelin immunoreactivity in the adult rat dentate gyrus and increased Dab1 expression in hilar-ectopic neuroblasts. Exogenous Reelin increased detachment of chain-migrating neuroblasts in dentate gyrus explants, and blockade of Reelin signaling increased chain migration. These findings suggest that Reelin modulates DGC progenitor migration to maintain normal DGC integration in the neonatal and adult mammalian dentate gyrus. Loss of Reelin expression in the epileptic adult hippocampus, moreover, likely contributes to ectopic chain migration and aberrant integration of newborn DGCs.
