Can Zhong - Academia.edu (original) (raw)
Papers by Can Zhong
Biochemical Journal, Jul 1, 2003
Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either... more Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent nutrient-sensing pathways converge on the same set of genomic cis -elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides -68 to -60 and the NSRE-2 core sequence is within nucleotides -48 to -43. Through insertion or deletion of 5-10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.
The Journal of Biological Chemistry, Oct 29, 1999
The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both a... more The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.
Topics in Current Genetics, 2004
Dietary protein is critical to mammalian nutrition and on a cellular level this translates into a... more Dietary protein is critical to mammalian nutrition and on a cellular level this translates into amino acid availability. Cells monitor amino acids and respond with changes in cellular processes, including gene transcription. Thus, amino acids serve as signal molecules to transmit the nutritional status of the organism to individual cells. Using two target genes, CHOP and asparagine synthetase, this chapter
Proceedings of the 10th World Congress on Intelligent Control and Automation, 2012
ABSTRACT This paper specifically describes the design of hardware and software of a digital pulse... more ABSTRACT This paper specifically describes the design of hardware and software of a digital pulse oximeter based on ARM core embedded system and light-frequency converter. In this study, a light-frequency converter instead of a photodiode was adopted to receive the light transmitting, which greatly simplified the design of hardware. Besides, traditional methods of calculate frequency of rectangle signal were improved and an algorithm with stable sampling rate and high resolution was proposed. Based on the filtering and detecting methods of the pulse signal, the baseline drift and the noise on high frequency were successfully removed by applying integer coefficient filter. A detecting algorithm based on difference and threshold was proposed, and the future points of the signal can be extracted effectively. The result shows that this oximeter can overcome usual noise, the software is very flexible and simple in structure.
The Biochemical journal, Jan 15, 2008
A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivatio... more A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and ...
Neurocomputing, 2015
Fundamental matrix estimation has been studied extensively in the area of computer vision and pre... more Fundamental matrix estimation has been studied extensively in the area of computer vision and previously proposed techniques include those that only use feature points. In this study, we propose a new technique for calculating the fundamental matrix combined with feature lines, which is based on the epipolar geometry of horizontal and vertical feature lines. First, a method for parameterizing the fundamental matrix is introduced, where the camera orientation elements and relative orientation elements are used as the parameters of the fundamental matrix, and the equivalent relationships are deduced based on the horizontal and vertical feature lines. Next, the feature lines are used as the interior points by the RANSAC algorithm to search for the optimal feature point subset, before determining the weight of each factor using the M-estimators algorithm and building a unified adjustment model to estimate the fundamental matrix. The experimental results obtained using simulated images and real images demonstrate that the proposed approach is feasible in practice and it can greatly reduce the dependency on feature points in the traditional method, while the introduction of feature lines can improve the accuracy and stability of the results to some extent.
Nucleic Acids Research, 2008
It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) t... more It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.
Journal of Biological Chemistry, 2000
The human asparagine synthetase (AS) gene is transcriptionally regulated by amino acid deprivatio... more The human asparagine synthetase (AS) gene is transcriptionally regulated by amino acid deprivation (amino acid response, AAR) and the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway. The results reported here document the novel observation that induction of the AS gene by the AAR and ERSR pathways occurs via the same set of genomic elements. Data supporting this conclusion include transient transfection of AS promoter/ reporter gene constructs that illustrate that the transcriptional control elements used by both pathways are contained with nucleotides ؊111 to ؊34 of the AS promoter. In vivo footprinting analysis of this region identified six specific protein-binding sites. Within two of these sites, altered footprinting was observed following amino acid or glucose deprivation, but the patterns were identical for both the AAR and the ERSR pathway. Site-directed mutation of individual nucleotides within these two binding sites confirmed their importance for regulated transcription, and none of the mutations resulted in loss of response of only one pathway. Neither of these two sites corresponds to a recently identified ERSR cis-element, nor do they contain consensus sequences for known transcription factors. Collectively, the data document that there are at least two independent transcriptional mechanisms for gene activation by the ERSR pathway, one of which terminates at the same genomic elements used by the AAR pathway.
Journal of Biological Chemistry, 2002
A promoter element called the amino acid response element (AARE), which is essential for the indu... more A promoter element called the amino acid response element (AARE), which is essential for the induction of CHOP (a CCAAT/enhancer-binding protein-related gene) transcription by amino acid depletion, has been previously characterized. Conversely, the human asparagine synthetase (AS) promoter contains two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and NSRE-2) that are required to activate the gene by either amino acid deprivation or the endoplasmic reticulum stress response. The results reported here document the comparison between CHOP and AS transcriptional control elements used by the amino acid pathway. We first establish that the AS NSRE-1 sequence shares nucleotide sequence and functional similarities with the CHOP AARE. However, we demonstrate that the CHOP AARE can function independently, whereas AS NSRE-1 is functionally weak by itself and instead requires the presence of NSRE-2. Furthermore, AS NSRE-2 can confer endoplasmic reticulum stress responsiveness to the CHOP AARE. Using activating transcription factor-2-deficient mouse embryonic fibroblasts, we also show that lack of this transcription factor does not abolish the amino acid inducibility of AS transcription, but this transcription factor is necessary to obtain the full AS response to amino acid starvation. Collectively, these results document that there are significant differences in the molecular mechanisms involved in the transcriptional activation of CHOP and AS by amino acid limitation.
Journal of Biological Chemistry, 2008
The mammalian amino acid response (AAR) pathway is upregulated by protein or amino acid depletion... more The mammalian amino acid response (AAR) pathway is upregulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of the translation initiation factor eIF2␣ (eukaryotic initiation factor 2␣), and, through subsequent translational control, enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation in HepG2 human hepatoma cells by PD98059 or U0126 blocked the increased phosphorylation of eIF2␣ and ATF4 synthesis triggered by amino acid limitation, showing that the AAR requires activation of the MEK-ERK pathway. Inhibitors of the JNK or p38 MAPK pathways were ineffective. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by overexpression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity and eIF2␣ phosphorylation. Inhibition of protein phosphatase 1 action on phospho-eIF2␣ by knockdown of GADD34 did not block the sensitivity to PD98059, suggesting that MEK functions to enhance GCN2-dependent eIF2␣ phosphorylation rather than suppressing dephosphorylation. Collectively, these results document a critical interdependence between the MEK-ERK MAPK signaling pathway and the amino acid stress-activated pathway.
![Research paper thumbnail of Erratum to “Controllable nanogap fabrication on microchip by chronopotentiometry” [Electrochimica Acta 50 (2005) 3041–3047]](https://attachments.academia-assets.com/46046160/thumbnails/1.jpg)
](Electrochimica Acta, 2006
The publisher regrets that "15 mM KAu(CN) 2 with 0.3 M K 3 C 6 H 5 O 7 and 0.3 M KH 2 PO 4 " in p... more The publisher regrets that "15 mM KAu(CN) 2 with 0.3 M K 3 C 6 H 5 O 7 and 0.3 M KH 2 PO 4 " in page 3043 should be corrected to "15 M KAu(CN) 2 with 0.3 mM K 3 C 6 H 5 O 7 and 0.3 mM KH 2 PO 4 " and " (b)" in page 3044 should be corrected to " ".
Biochemical Journal, 2008
A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivatio... more A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPbeta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPbeta protein was phosphorylated on Thr(235) and the phospho-C/EBPbeta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPbeta-deficient MEFs (mouse embryonic fibroblasts) or C/EBPbeta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPbeta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPbeta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.
Biochemical Journal, 2003
Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either... more Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent nutrient-sensing pathways converge on the same set of genomic cis -elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides -68 to -60 and the NSRE-2 core sequence is within nucleotides -48 to -43. Through insertion or deletion of 5-10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.
Biochemical Journal, 2006
The Journal of biological chemistry, Jan 2, 2004
CCAAT/enhancer-binding protein beta (C/EBPbeta) is a member of the bZIP family of transcription f... more CCAAT/enhancer-binding protein beta (C/EBPbeta) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBPbeta gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBPbeta genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBPbeta promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3'-untranslated region of the C/EBPbeta mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt +1614-1621 (5'-TGACGCAA-3') is responsible for activation of the C/EBPbeta gene. Electrophoresis mobility sh...
Biochemical Journal, Jul 1, 2003
Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either... more Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent nutrient-sensing pathways converge on the same set of genomic cis -elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides -68 to -60 and the NSRE-2 core sequence is within nucleotides -48 to -43. Through insertion or deletion of 5-10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.
The Journal of Biological Chemistry, Oct 29, 1999
The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both a... more The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.
Topics in Current Genetics, 2004
Dietary protein is critical to mammalian nutrition and on a cellular level this translates into a... more Dietary protein is critical to mammalian nutrition and on a cellular level this translates into amino acid availability. Cells monitor amino acids and respond with changes in cellular processes, including gene transcription. Thus, amino acids serve as signal molecules to transmit the nutritional status of the organism to individual cells. Using two target genes, CHOP and asparagine synthetase, this chapter
Proceedings of the 10th World Congress on Intelligent Control and Automation, 2012
ABSTRACT This paper specifically describes the design of hardware and software of a digital pulse... more ABSTRACT This paper specifically describes the design of hardware and software of a digital pulse oximeter based on ARM core embedded system and light-frequency converter. In this study, a light-frequency converter instead of a photodiode was adopted to receive the light transmitting, which greatly simplified the design of hardware. Besides, traditional methods of calculate frequency of rectangle signal were improved and an algorithm with stable sampling rate and high resolution was proposed. Based on the filtering and detecting methods of the pulse signal, the baseline drift and the noise on high frequency were successfully removed by applying integer coefficient filter. A detecting algorithm based on difference and threshold was proposed, and the future points of the signal can be extracted effectively. The result shows that this oximeter can overcome usual noise, the software is very flexible and simple in structure.
The Biochemical journal, Jan 15, 2008
A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivatio... more A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and ...
Neurocomputing, 2015
Fundamental matrix estimation has been studied extensively in the area of computer vision and pre... more Fundamental matrix estimation has been studied extensively in the area of computer vision and previously proposed techniques include those that only use feature points. In this study, we propose a new technique for calculating the fundamental matrix combined with feature lines, which is based on the epipolar geometry of horizontal and vertical feature lines. First, a method for parameterizing the fundamental matrix is introduced, where the camera orientation elements and relative orientation elements are used as the parameters of the fundamental matrix, and the equivalent relationships are deduced based on the horizontal and vertical feature lines. Next, the feature lines are used as the interior points by the RANSAC algorithm to search for the optimal feature point subset, before determining the weight of each factor using the M-estimators algorithm and building a unified adjustment model to estimate the fundamental matrix. The experimental results obtained using simulated images and real images demonstrate that the proposed approach is feasible in practice and it can greatly reduce the dependency on feature points in the traditional method, while the introduction of feature lines can improve the accuracy and stability of the results to some extent.
Nucleic Acids Research, 2008
It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) t... more It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.
Journal of Biological Chemistry, 2000
The human asparagine synthetase (AS) gene is transcriptionally regulated by amino acid deprivatio... more The human asparagine synthetase (AS) gene is transcriptionally regulated by amino acid deprivation (amino acid response, AAR) and the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway. The results reported here document the novel observation that induction of the AS gene by the AAR and ERSR pathways occurs via the same set of genomic elements. Data supporting this conclusion include transient transfection of AS promoter/ reporter gene constructs that illustrate that the transcriptional control elements used by both pathways are contained with nucleotides ؊111 to ؊34 of the AS promoter. In vivo footprinting analysis of this region identified six specific protein-binding sites. Within two of these sites, altered footprinting was observed following amino acid or glucose deprivation, but the patterns were identical for both the AAR and the ERSR pathway. Site-directed mutation of individual nucleotides within these two binding sites confirmed their importance for regulated transcription, and none of the mutations resulted in loss of response of only one pathway. Neither of these two sites corresponds to a recently identified ERSR cis-element, nor do they contain consensus sequences for known transcription factors. Collectively, the data document that there are at least two independent transcriptional mechanisms for gene activation by the ERSR pathway, one of which terminates at the same genomic elements used by the AAR pathway.
Journal of Biological Chemistry, 2002
A promoter element called the amino acid response element (AARE), which is essential for the indu... more A promoter element called the amino acid response element (AARE), which is essential for the induction of CHOP (a CCAAT/enhancer-binding protein-related gene) transcription by amino acid depletion, has been previously characterized. Conversely, the human asparagine synthetase (AS) promoter contains two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and NSRE-2) that are required to activate the gene by either amino acid deprivation or the endoplasmic reticulum stress response. The results reported here document the comparison between CHOP and AS transcriptional control elements used by the amino acid pathway. We first establish that the AS NSRE-1 sequence shares nucleotide sequence and functional similarities with the CHOP AARE. However, we demonstrate that the CHOP AARE can function independently, whereas AS NSRE-1 is functionally weak by itself and instead requires the presence of NSRE-2. Furthermore, AS NSRE-2 can confer endoplasmic reticulum stress responsiveness to the CHOP AARE. Using activating transcription factor-2-deficient mouse embryonic fibroblasts, we also show that lack of this transcription factor does not abolish the amino acid inducibility of AS transcription, but this transcription factor is necessary to obtain the full AS response to amino acid starvation. Collectively, these results document that there are significant differences in the molecular mechanisms involved in the transcriptional activation of CHOP and AS by amino acid limitation.
Journal of Biological Chemistry, 2008
The mammalian amino acid response (AAR) pathway is upregulated by protein or amino acid depletion... more The mammalian amino acid response (AAR) pathway is upregulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of the translation initiation factor eIF2␣ (eukaryotic initiation factor 2␣), and, through subsequent translational control, enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation in HepG2 human hepatoma cells by PD98059 or U0126 blocked the increased phosphorylation of eIF2␣ and ATF4 synthesis triggered by amino acid limitation, showing that the AAR requires activation of the MEK-ERK pathway. Inhibitors of the JNK or p38 MAPK pathways were ineffective. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by overexpression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity and eIF2␣ phosphorylation. Inhibition of protein phosphatase 1 action on phospho-eIF2␣ by knockdown of GADD34 did not block the sensitivity to PD98059, suggesting that MEK functions to enhance GCN2-dependent eIF2␣ phosphorylation rather than suppressing dephosphorylation. Collectively, these results document a critical interdependence between the MEK-ERK MAPK signaling pathway and the amino acid stress-activated pathway.
Electrochimica Acta, 2006
The publisher regrets that "15 mM KAu(CN) 2 with 0.3 M K 3 C 6 H 5 O 7 and 0.3 M KH 2 PO 4 " in p... more The publisher regrets that "15 mM KAu(CN) 2 with 0.3 M K 3 C 6 H 5 O 7 and 0.3 M KH 2 PO 4 " in page 3043 should be corrected to "15 M KAu(CN) 2 with 0.3 mM K 3 C 6 H 5 O 7 and 0.3 mM KH 2 PO 4 " and " (b)" in page 3044 should be corrected to " ".
Biochemical Journal, 2008
A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivatio... more A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPbeta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPbeta protein was phosphorylated on Thr(235) and the phospho-C/EBPbeta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPbeta-deficient MEFs (mouse embryonic fibroblasts) or C/EBPbeta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPbeta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPbeta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.
Biochemical Journal, 2003
Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either... more Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent nutrient-sensing pathways converge on the same set of genomic cis -elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides -68 to -60 and the NSRE-2 core sequence is within nucleotides -48 to -43. Through insertion or deletion of 5-10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.
Biochemical Journal, 2006
The Journal of biological chemistry, Jan 2, 2004
CCAAT/enhancer-binding protein beta (C/EBPbeta) is a member of the bZIP family of transcription f... more CCAAT/enhancer-binding protein beta (C/EBPbeta) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBPbeta gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBPbeta genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBPbeta promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3'-untranslated region of the C/EBPbeta mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt +1614-1621 (5'-TGACGCAA-3') is responsible for activation of the C/EBPbeta gene. Electrophoresis mobility sh...