scholarly journals Direct transcriptional stimulation of the ornithine decarboxylase gene by Fos in PC12 cells but not in fibroblasts.

1993 ◽  
Vol 13 (8) ◽  
pp. 4657-4669 ◽  
Author(s):  
C Wrighton ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Pc12 Cells ◽  
Ornithine Decarboxylase ◽  
Transcription Initiation ◽  
Prolonged Exposure ◽  
Gene Activation ◽  
Neuronal Cell ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Stimulation Of

We have established rat PC12 pheochromocytoma cell lines stably expressing the estrogen-activatable transcription factor FosER to identify genes that can be regulated by c-Fos in this neuronal cell type. Induction of ectopic c-Fos activity in PC12 cells increased the mRNA levels of the ornithine decarboxylase (ODC) and tyrosine hydroxylase genes with similar kinetics and to the same maximal level as nerve growth factor treatment. In both cases the rate of transcription initiation was increased. Induction of the ODC gene occurred even in the absence of protein synthesis, indicating direct regulation by FosER. ODC expression, however, was not induced by a mutant FosER protein containing a proline insertion in the basic region of the c-Fos moiety, demonstrating the requirement for a functional DNA-binding domain. These data show that FosER, and by extrapolation c-Fos, can directly activate transcription of the endogenous ODC gene in PC12 cells by binding to cis-regulatory sequences. Activation of the ODC gene was unexpectedly transient, as transcripts returned to the basal level after prolonged exposure of PC12 cells to FosER activity. Furthermore, ODC transcription was not at all induced by FosER in rat fibroblasts. To account for this cell-specific action of FosER, we propose that stimulation of the ODC gene by FosER requires either (i) cooperation with another transcription factor(s) or (ii) a specific pattern of modification which is present in PC12 cells but not in otherwise unstimulated fibroblasts. One or both of these mechanisms may be employed by cells to achieve selective gene activation in response to apparently stereotyped induction of c-fos.

Direct transcriptional stimulation of the ornithine decarboxylase gene by Fos in PC12 cells but not in fibroblasts

1993 ◽  
Vol 13 (8) ◽  
pp. 4657-4669
Author(s):  
C Wrighton ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Pc12 Cells ◽  
Ornithine Decarboxylase ◽  
Transcription Initiation ◽  
Prolonged Exposure ◽  
Gene Activation ◽  
Neuronal Cell ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Stimulation Of

We have established rat PC12 pheochromocytoma cell lines stably expressing the estrogen-activatable transcription factor FosER to identify genes that can be regulated by c-Fos in this neuronal cell type. Induction of ectopic c-Fos activity in PC12 cells increased the mRNA levels of the ornithine decarboxylase (ODC) and tyrosine hydroxylase genes with similar kinetics and to the same maximal level as nerve growth factor treatment. In both cases the rate of transcription initiation was increased. Induction of the ODC gene occurred even in the absence of protein synthesis, indicating direct regulation by FosER. ODC expression, however, was not induced by a mutant FosER protein containing a proline insertion in the basic region of the c-Fos moiety, demonstrating the requirement for a functional DNA-binding domain. These data show that FosER, and by extrapolation c-Fos, can directly activate transcription of the endogenous ODC gene in PC12 cells by binding to cis-regulatory sequences. Activation of the ODC gene was unexpectedly transient, as transcripts returned to the basal level after prolonged exposure of PC12 cells to FosER activity. Furthermore, ODC transcription was not at all induced by FosER in rat fibroblasts. To account for this cell-specific action of FosER, we propose that stimulation of the ODC gene by FosER requires either (i) cooperation with another transcription factor(s) or (ii) a specific pattern of modification which is present in PC12 cells but not in otherwise unstimulated fibroblasts. One or both of these mechanisms may be employed by cells to achieve selective gene activation in response to apparently stereotyped induction of c-fos.

scholarly journals Protective Effect of Psoralea corylifolia L. Seed Extract against Palmitate-Induced Neuronal Apoptosis in PC12 Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Yunkyoung Lee ◽  
Hee-Sook Jun ◽  
Yoon Sin Oh
Keyword(s):  
Pc12 Cells ◽  
Molecular Mechanisms ◽  
Neuronal Cell ◽  
Marker Genes ◽  
Heme Oxygenase 1 ◽  
Mrna Levels ◽  
Protective Effects ◽  
Psoralea Corylifolia ◽  
Antioxidant Genes ◽  
Bax Protein

The extract of Psoralea corylifolia seeds (PCE) has been widely used as a herbal medicine because of its beneficial effect on human health. In this study, we investigated the protective effects and molecular mechanisms of PCE on palmitate- (PA-) induced toxicity in PC12 cells, a neuron-like cell line. PCE significantly increased cell viability in PA-treated PC12 cells and showed antiapoptotic effects, as evidenced by decreased expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, and bax protein as well as increased expression of bcl-2 protein. In addition, PCE treatment reduced PA-induced reactive oxygen species production and upregulated mRNA levels of antioxidant genes such as nuclear factor (erythroid-derived 2)-like 2 and heme oxygenase 1. Moreover, PCE treatment recovered the expression of autophagy marker genes such as beclin-1 and p62, which was decreased by PA treatment. Treatment with isopsoralen, one of the major components of PCE extract, also recovered the expression of autophagy marker genes and reduced PA-induced apoptosis. In conclusion, PCE exerts protective effects against lipotoxicity via its antioxidant function, and this effect is mediated by activation of autophagy. PCE might be a potential pharmacological agent to protect against neuronal cell injury caused by oxidative stress or lipotoxicity.

scholarly journals Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells

2015 ◽  
Vol 37 (5) ◽  
pp. 1750-1758 ◽  
Author(s):  
Eleni Stamoula ◽  
Theofanis Vavilis ◽  
Eleni Aggelidou ◽  
Aikaterini Kaidoglou ◽  
Angeliki Cheva ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Cell Fate ◽  
Low Dose ◽  
Mrna Level ◽  
Neuronal Cell ◽  
Cellular Level ◽  
Mrna Levels ◽  
Protein Levels ◽  
Low Concentrations ◽  
Discrete Effect

Background/Aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

scholarly journals Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

2016 ◽  
Author(s):  
David B. Nyer ◽  
Daniel Vargas ◽  
Caroline Hom ◽  
Karmella A. Haynes
Keyword(s):  
Transcription Factor ◽  
Histone Modifications ◽  
Transcription Initiation ◽  
Gene Activation ◽  
Promoter Regions ◽  
Protein Protein Interaction ◽  
Synthetic Proteins ◽  
Transcription Initiation Complex ◽  
Endogenous Genes ◽  
Chromatin Profiling

ABSTRACTChromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously we reported the first synthetic transcriptional activator, the "Polycomb-based transcription factor" (PcTF), that reads histone modifications through a protein-protein interaction between the PCD motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of PcTF. Transcriptome and chromatin profiling revealed PcTF-sensitive promoter regions marked by proximal PcTF and distal H3K27me3 binding. These results illuminate a mechanism in which PcTF interactions bridge epigenetic marks with the transcription initiation complex. In three cancer-derived human cell lines tested here, many PcTF-sensitive genes encode developmental regulators and tumor suppressors. Thus, PcTF represents a powerful new fusion-protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation.

The SCL-LMO2 Interaction Nucleates Hematopoietic Transcription Factor Complexes: Coupling Protein Stability and Complex Assembly.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 779-779
Author(s):  
Marie-Claude Sincennes ◽  
Eric Lecuyer ◽  
Simon Lariviere ◽  
Andre Haman ◽  
Rachid Lahlil ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Stability ◽  
Cell Line ◽  
Half Life ◽  
Gene Activation ◽  
Hematopoietic Cells ◽  
Mrna Levels ◽  
Protein Levels ◽  
Transcription Factor Complexes

Abstract The differentiation of hematopoietic cells is tightly controlled by transcription factor complexes, composed of hemato-specific and ubiquitous proteins. The bHLH factor SCL and the LIM-only protein LMO2 are central components of transcription factor complexes and are essential for hematopoiesis. However, the mechanism regulating the assembly of SCL-complexes is unknown. Here we show that SCL, in contrast to LMO, GATA and E proteins, cannot be replaced by other members of its family in hematopoietic gene transactivation and in gel shift assays. Furthermore, we show by GST pull-down assays and by co-immunoprecipitation that interaction with LMO2 is a unique property of SCL, as the neurogenic bHLH NSCL1 related to SCL cannot bind LMO2. By generating SCL-NSCL1 chimeras, and by phylogenetic alignment, we identified the SCL interface that confers transcriptional specificity to the complex. Strikingly, this interface is also necessary for the interaction with LMO2. In contrast with the wild type SCL, the mutant without this interface is not able to enhance erythroid differentiation when overexpressed in hematopoietic cells, as assessed by glycophorin A gene activation, benzidine staining and methylcellulose cultures. Interestingly, we also demonstrate in vivo and in vitro that LMO2 protein levels are greatly increased in the presence of SCL, while mRNA levels remain constant. When the SCL interface described above is mutated, LMO2 protein level is no longer increased, suggesting that the accumulation of LMO2 is mediated by a direct interaction with SCL. In primary hematopoietic cells, when SCL protein levels are genetically reduced by LacZ insertion into one allele in the SCL locus, we observe a dramatic decrease in LMO2 protein levels. In addition, in the TF-1 hematopoietic cell line, most of the LMO2 protein (90%) is found associated with SCL and/or Ldb1, suggesting that free LMO2 is rapidly degraded. Thus, SCL levels determine LMO2 levels in hematopoietic cells. Next, we provide direct evidence that LMO2 is a target for proteasomal degradation. First, we show that LMO2 is ubiquitinated in vivo, by GST purification. Second, by using the ts20 cell line expressing a temperature-sensitive ubiquitin-activating E1 enzyme, we show that LMO2 degradation requires a functional ubiquitin conjugation system, since LMO2 is not degraded when E1 is inactive. Third, we show that the half-life of LMO2 is very short, and it can be increased with the proteasome inhibitor MG132. Finally, a similar increase in LMO2 half-life can be observed when SCL is co-expressed with LMO2. These data indicate that SCL stabilizes LMO2, which is otherwise rapidly degraded by the ubiquitin-proteasome pathway in absence of its interacting partners. Taken together, our results strongly suggest that SCL, by binding and stabilizing LMO2, is a critical determinant of the hematopoietic transcriptional specificity. The interaction between SCL and LMO2 is an essential nucleation step for the assembly of SCL-complexes on DNA, where the regulation of LMO2 levels appears to be the rate-limiting step. We propose that protein stability is a new mechanism of regulation in the formation of SCL complexes, required for proper gene activation during eryhtroid differentiation.

scholarly journals Transcriptional Responses in Recovery from Stroke

Stroke ◽  
2001 ◽  
Vol 32 (suppl_1) ◽  
pp. 316-316
Author(s):  
Sandra Bolanos ◽  
Hiroshi Saito ◽  
John Papaconstantinou ◽  
Thomas A Kent
Keyword(s):  
Pc12 Cells ◽  
Synapse Formation ◽  
Neuronal Cell ◽  
Long Term Potentiation ◽  
Signal Molecules ◽  
Cell Transplant ◽  
Transcriptional Responses ◽  
Infarct Region ◽  
Stimulation Of

1 Multiple lines of evidence support synaptic reorganization of the brain after stroke and a role in functional recovery. Molecular, pharmacological, especially norardrenergic, and behavioral methods have shown promise in enhancing recovery in animal or clinical studies. However, the mechanisms underlying stimulation of new synapses remain largely unknown, information that is critical to optimize interventions, including stem cell transplant, drug therapy or other approaches. We investigated two potential such mechanisms in a rat model of middle cerebral artery occlusion (MCAO) in which we have previously shown robust new expression of the pre-synaptic vesicle protein synaptophysin in the peri-infarct and contralateral homotopic regions. One candidate signal for stimulation of new synaptic formation is the polysialated form of neuronal cell adhesion molecules (PSCAM) that is expressed during synaptic development. Immunostaining for PSCAM after MCAO and recovery failed to demonstrate expression. The possibility that signal molecules potentially released following ischemia, may be involved in synaptic generation analogous to long term potentiation (LTP), was next investigated. The C/EBP (CCAAT enhancer binding protein) family of transcription factors is an important intermediary for glutamate stimulation of synapses in LTP. After distal MCAO in rats, we found dramatic expression of the C/EBP α subtype in the peri-infarct region at 3 days, and expression by Western blot of a 30 kD C/EBP α isoform in cultured PC12 cells induced to differentiate into neurite and synapses. We suggest the possibility that C/EBP, stimulated by biochemical events in the peri-infarct region, is a potential signal for new synapses following stroke. We are in the process of assessing the effect of overexpression of this isoform on synapse formation in cultured PC12 cells. C/EBP α and other signals may provide targets for intervention to enhance expression. The importance of these results as related to the effects of glutamate also support our previous finding that glutamate-blockade, although limiting infarct size, may also interfere with synapse formation in the long term (Bolanos & Kent, JCBF & Met Suppl, 1999).

scholarly journals Egr3/Pilot, a zinc finger transcription factor, is rapidly regulated by activity in brain neurons and colocalizes with Egr1/zif268.

1994 ◽  
Vol 1 (2) ◽  
pp. 140-152
Author(s):  
K Yamagata ◽  
W E Kaufmann ◽  
A Lanahan ◽  
M Papapavlou ◽  
C A Barnes ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Granule Cells ◽  
Gene Activation ◽  
Receptor Activation ◽  
Monocular Deprivation ◽  
Synaptic Activity ◽  
Mrna Levels ◽  
Zinc Finger Transcription Factor

Programs of gene activation may underlie long-term adaptive cellular responses to extracellular ligands. We have used a differential cDNA cloning strategy to identify genes that are strongly induced by excitatory stimuli in the adult rat hippocampus. Here, we report the rat cDNA sequence of a zinc-finger transcription factor, Egr3/Pilot, and characterize its regulated mRNA expression in brain. Egr3 mRNA is rapidly and transiently induced in neurons of the hippocampus and cortex by electroconvulsive seizure. mRNA levels peak 2 hr after the seizure and remain elevated for as long as 8 hr. Egr3 mRNA is also rapidly induced in granule cells of the dentate gyrus by synaptic NMDA receptor activation elicited by patterned stimulation of the perforant pathway and by drugs that alter dopamine neurotransmission in the striatum. Basal levels of Egr3 mRNA in the cortex appear to be driven by natural synaptic activity because monocular deprivation rapidly decreases Egr3 mRNA in the deafferented visual cortex. Aspects of the protein structure, sequence-specific DNA binding, transcriptional activity, and regulation of Egr3 are highly similar to another zinc-finger transcription factor, Egr1/zif268. Moreover, we demonstrate colocalization of Egr3 and zif268 mRNAs in neurons of normal and stimulated cortex. Our studies suggest that interactions between these coregulated transcription factors may be important in defining long-term, neuroplastic responses.

scholarly journals Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon.

1990 ◽  
Vol 10 (7) ◽  
pp. 3468-3482 ◽  
Author(s):  
W Nikovits ◽  
J H Mar ◽  
C P Ordahl
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Troponin I ◽  
Specific Activity ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Deletion Analysis ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Cat Gene

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.

Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon

1990 ◽  
Vol 10 (7) ◽  
pp. 3468-3482
Author(s):  
W Nikovits ◽  
J H Mar ◽  
C P Ordahl
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Troponin I ◽  
Specific Activity ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Deletion Analysis ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Cat Gene

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

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