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Murray L. Whitelaw - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterisation of rare loss-of-function NPAS3 and NPAS4 variants identified in individuals with neurodevelopmental disorders.
Scientific reports, 2021Co-Authors: Joseph J Rossi, Daniel J. Peet, Murray L. Whitelaw, Jill A Rosenfeld, Katie M Chan, Haley Streff, Victoria Nankivell, David C. BerstenAbstract:Aberrations in the excitatory/inhibitory balance within the brain have been associated with both intellectual disability (ID) and schizophrenia (SZ). The bHLH-PAS transcription factors NPAS3 and NPAS4 have been implicated in controlling the excitatory/inhibitory balance, and targeted disruption of either gene in mice results in a phenotype resembling ID and SZ. However, there are few human variants in NPAS3 and none in NPAS4 that have been associated with schizophrenia or neurodevelopmental disorders. From a clinical exome sequencing database we identified three NPAS3 variants and four NPAS4 variants that could potentially disrupt protein function in individuals with either developmental delay or ID. The transcriptional activity of the variants when partnered with either ARNT or ARNT2 was assessed by reporter gene activity and it was found that variants which truncated the NPAS3/4 protein resulted in a complete loss of transcriptional activity. The ability of loss-of-function variants to heterodimerise with neuronally enriched partner protein ARNT2 was then determined by co-immunoprecipitation experiments. It was determined that the mechanism for the observed loss of function was the inability of the truncated NPAS3/4 protein to heterodimerise with ARNT2. This further establishes NPAS3 and NPAS4 as candidate neurodevelopmental disorder genes.
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Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2.
The Biochemical journal, 2014Co-Authors: Adrienne E. Sullivan, Daniel J. Peet, John B. Bruning, Anne Raimondo, Tanja A. Schwab, Philippe Froguel, I. Sadaf Farooqi, Murray L. WhitelawAbstract:The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hot-spot’ in SIM1 that is critical for protein function.
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Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function
PloS one, 2014Co-Authors: David C. Bersten, Daniel J. Peet, John B. Bruning, Murray L. WhitelawAbstract:Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.
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human variants in the neuronal basic helix loop helix per arnt sim bhlh pas transcription factor complex npas4 ARNT2 disrupt function
PLOS ONE, 2014Co-Authors: David C. Bersten, Daniel J. Peet, John B. Bruning, Murray L. WhitelawAbstract:Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.
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reciprocal regulation of the basic helix loop helix per arnt sim partner proteins arnt and ARNT2 during neuronal differentiation
Nucleic Acids Research, 2013Co-Authors: Nan Hao, Veronica L. D. Bhakti, Daniel J. Peet, Murray L. WhitelawAbstract:Basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) transcription factors function broadly in development, homeostasis and stress response. Active bHLH/PAS heterodimers consist of a ubiquitous signal-regulated subunit (e.g., hypoxia-inducible factors, HIF-1α/2α/3α; the aryl hydrocarbon receptor, AhR) or tissue-restricted subunit (e.g., NPAS1/3/4, Single Minded 1/2), paired with a general partner protein, aryl hydrocarbon receptor nuclear translocator (Arnt or ARNT2). We have investigated regulation of the neuron-enriched Arnt paralogue, ARNT2. We find high Arnt/ARNT2 ratios in P19 embryonic carcinoma cells and ES cells are dramatically reversed to high ARNT2/Arnt on neuronal differentiation. mRNA half-lives of Arnt and ARNT2 remain similar in both parent and neuronal differentiated cells. The GC-rich ARNT2 promoter, while heavily methylated in Arnt only expressing hepatoma cells, is methylation free in P19 and ES cells, where it is bivalent with respect to active H3K4me3 and repressive H3K27me3 histone marks. Typical of a 'transcription poised' developmental gene, H3K27me3 repressive marks are removed from ARNT2 during neuronal differentiation. Our data are consistent with a switch to predominant ARNT2 expression in neurons to allow specific functions of neuronal bHLH/PAS factors and/or to avoid neuronal bHLH/PAS factors from interfering with AhR/Arnt signalling.
Daniel J. Peet - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterisation of rare loss-of-function NPAS3 and NPAS4 variants identified in individuals with neurodevelopmental disorders.
Scientific reports, 2021Co-Authors: Joseph J Rossi, Daniel J. Peet, Murray L. Whitelaw, Jill A Rosenfeld, Katie M Chan, Haley Streff, Victoria Nankivell, David C. BerstenAbstract:Aberrations in the excitatory/inhibitory balance within the brain have been associated with both intellectual disability (ID) and schizophrenia (SZ). The bHLH-PAS transcription factors NPAS3 and NPAS4 have been implicated in controlling the excitatory/inhibitory balance, and targeted disruption of either gene in mice results in a phenotype resembling ID and SZ. However, there are few human variants in NPAS3 and none in NPAS4 that have been associated with schizophrenia or neurodevelopmental disorders. From a clinical exome sequencing database we identified three NPAS3 variants and four NPAS4 variants that could potentially disrupt protein function in individuals with either developmental delay or ID. The transcriptional activity of the variants when partnered with either ARNT or ARNT2 was assessed by reporter gene activity and it was found that variants which truncated the NPAS3/4 protein resulted in a complete loss of transcriptional activity. The ability of loss-of-function variants to heterodimerise with neuronally enriched partner protein ARNT2 was then determined by co-immunoprecipitation experiments. It was determined that the mechanism for the observed loss of function was the inability of the truncated NPAS3/4 protein to heterodimerise with ARNT2. This further establishes NPAS3 and NPAS4 as candidate neurodevelopmental disorder genes.
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Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2.
The Biochemical journal, 2014Co-Authors: Adrienne E. Sullivan, Daniel J. Peet, John B. Bruning, Anne Raimondo, Tanja A. Schwab, Philippe Froguel, I. Sadaf Farooqi, Murray L. WhitelawAbstract:The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hot-spot’ in SIM1 that is critical for protein function.
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Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function
PloS one, 2014Co-Authors: David C. Bersten, Daniel J. Peet, John B. Bruning, Murray L. WhitelawAbstract:Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.
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human variants in the neuronal basic helix loop helix per arnt sim bhlh pas transcription factor complex npas4 ARNT2 disrupt function
PLOS ONE, 2014Co-Authors: David C. Bersten, Daniel J. Peet, John B. Bruning, Murray L. WhitelawAbstract:Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.
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reciprocal regulation of the basic helix loop helix per arnt sim partner proteins arnt and ARNT2 during neuronal differentiation
Nucleic Acids Research, 2013Co-Authors: Nan Hao, Veronica L. D. Bhakti, Daniel J. Peet, Murray L. WhitelawAbstract:Basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) transcription factors function broadly in development, homeostasis and stress response. Active bHLH/PAS heterodimers consist of a ubiquitous signal-regulated subunit (e.g., hypoxia-inducible factors, HIF-1α/2α/3α; the aryl hydrocarbon receptor, AhR) or tissue-restricted subunit (e.g., NPAS1/3/4, Single Minded 1/2), paired with a general partner protein, aryl hydrocarbon receptor nuclear translocator (Arnt or ARNT2). We have investigated regulation of the neuron-enriched Arnt paralogue, ARNT2. We find high Arnt/ARNT2 ratios in P19 embryonic carcinoma cells and ES cells are dramatically reversed to high ARNT2/Arnt on neuronal differentiation. mRNA half-lives of Arnt and ARNT2 remain similar in both parent and neuronal differentiated cells. The GC-rich ARNT2 promoter, while heavily methylated in Arnt only expressing hepatoma cells, is methylation free in P19 and ES cells, where it is bivalent with respect to active H3K4me3 and repressive H3K27me3 histone marks. Typical of a 'transcription poised' developmental gene, H3K27me3 repressive marks are removed from ARNT2 during neuronal differentiation. Our data are consistent with a switch to predominant ARNT2 expression in neurons to allow specific functions of neuronal bHLH/PAS factors and/or to avoid neuronal bHLH/PAS factors from interfering with AhR/Arnt signalling.
Masahiko Nomura - One of the best experts on this subject based on the ideXlab platform.
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cdna cloning and tissue specific expression of a novel basic helix loop helix pas protein bmal1 and identification of alternatively spliced variants with alternative translation initiation site usage
Biochemical and Biophysical Research Communications, 1997Co-Authors: Masaaki Ikeda, Masahiko NomuraAbstract:Abstract Basic helix-loop-helix (bHLH)/PAS proteins, such as Sim, act as transcriptional factors, playing a critical role in the control of central nervous system (CNS) development. To isolate novel bHLH/PAS factors in the CNS an iterative search of a database for expressed sequence tags (ESTs) resulted in the location of several bHLH/PAS protein-like sequences. The rapid amplification of cDNA end (RACE) method was applied to isolate full-length cDNAs of these ESTs. Several 5′ and 3′ terminal sequences were isolated using primers derived from an EST from the human brain cDNA library. The predicted novel factor polypeptide had bHLH and PAS domains that were highly homologous with those of Ah receptor nuclear translocator (Arnt) and ARNT2. Combination of the isolated cDNA fragments revealed the existence of several alternatively spliced variants. The distribution of the novel bHLH/PAS factor message was analyzed by Northern blot hybridization. This detected only one transcript, which was 2.9 kb in size. Strong hybridization was found in the brain, skeletal muscle and heart. Expression of the novel bHLH/PAS factor, brain and muscle Arnt-like protein 1 (BMAL1), was different from that of Arnt and ARNT2, suggesting that BMAL1 has a different function in the CNS and muscle than Arnt and ARNT2.
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cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage.
Biochemical and biophysical research communications, 1997Co-Authors: Masaaki Ikeda, Masahiko NomuraAbstract:Abstract Basic helix-loop-helix (bHLH)/PAS proteins, such as Sim, act as transcriptional factors, playing a critical role in the control of central nervous system (CNS) development. To isolate novel bHLH/PAS factors in the CNS an iterative search of a database for expressed sequence tags (ESTs) resulted in the location of several bHLH/PAS protein-like sequences. The rapid amplification of cDNA end (RACE) method was applied to isolate full-length cDNAs of these ESTs. Several 5′ and 3′ terminal sequences were isolated using primers derived from an EST from the human brain cDNA library. The predicted novel factor polypeptide had bHLH and PAS domains that were highly homologous with those of Ah receptor nuclear translocator (Arnt) and ARNT2. Combination of the isolated cDNA fragments revealed the existence of several alternatively spliced variants. The distribution of the novel bHLH/PAS factor message was analyzed by Northern blot hybridization. This detected only one transcript, which was 2.9 kb in size. Strong hybridization was found in the brain, skeletal muscle and heart. Expression of the novel bHLH/PAS factor, brain and muscle Arnt-like protein 1 (BMAL1), was different from that of Arnt and ARNT2, suggesting that BMAL1 has a different function in the CNS and muscle than Arnt and ARNT2.
Jean-michel Arrang - One of the best experts on this subject based on the ideXlab platform.
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two splice variants of the hypoxia inducible factor hif 1α as potential dimerization partners of ARNT2 in neurons
European Journal of Neuroscience, 2000Co-Authors: Guillaume Drutel, Markus Kathmann, Anne Héron, Jean-charles Schwartz, Claude Gros, Séverine Macé, Jean-michel ArrangAbstract:The hypoxia-inducible factor (HIF-1alpha), a basic helix-loop-helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF-1alpha and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF-1alpha is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single-minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF-1alpha in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF-1alpha/ARNT1 complex, does not recognize the HIF-1-binding site of the hypoxia-induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF-1alpha and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF-1alpha, ARNT1 and ARNT2, downregulation of either HIF-1alpha or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.
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Two splice variants of the hypoxia‐inducible factor HIF‐1α as potential dimerization partners of ARNT2 in neurons
The European journal of neuroscience, 2000Co-Authors: Guillaume Drutel, Markus Kathmann, Anne Héron, Jean-charles Schwartz, Claude Gros, Séverine Macé, Jean-michel ArrangAbstract:The hypoxia-inducible factor (HIF-1alpha), a basic helix-loop-helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF-1alpha and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF-1alpha is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single-minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF-1alpha in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF-1alpha/ARNT1 complex, does not recognize the HIF-1-binding site of the hypoxia-induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF-1alpha and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF-1alpha, ARNT1 and ARNT2, downregulation of either HIF-1alpha or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.
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ARNT2, a transcription factor for brain neuron survival?
The European journal of neuroscience, 1999Co-Authors: Guillaume Drutel, Markus Kathmann, Anne Héron, Jean-charles Schwartz, Claude Gros, Séverine Macé, Michel Plotkine, Jean-michel ArrangAbstract:The processes responsible for the limited ability to divide and long survival of neurons are not well understood but may involve aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a recently identified protein, apparently belonging to the basic helix-loop-helix superfamily of transcription factors, which is expressed almost exclusively in brain during the whole lifetime. In agreement, we show, in the rat, that ARNT2 immunoreactivity could be observed only within nuclei of brain neurons and of dividing and neuronal PC12 cells, a localization consistent with a role in transcription regulation. Cell death elicited either by focal ischaemia in brain or oxidative stress in PC12 cells was largely preceded by an almost complete suppression of ARNT2 expression. In contrast, when PC12 cell cycle progression was impaired, ARNT2 expression was enhanced. Finally, the downregulation of ARNT2 levels induced by antisense oligonucleotides prevented PC12 cell proliferation and induced apoptosis. These observations support the hypothesis that ARNT2 is a neuronal transcription factor, regulating cell cycle progression and preventing cell death, whose sustained expression might ensure brain neuron survival.
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Cloning and Selective Expression in Brain and Kidney of ARNT2 Homologous to the Ah Receptor Nuclear Translocator (ARNT)
Biochemical and Biophysical Research Communications, 1996Co-Authors: Guillaume Drutel, Markus Kathmann, Anne Héron, Jean-charles Schwartz, Jean-michel ArrangAbstract:ARNT2, a new member of the basic-helix-loop-helix transcription factor family, was cloned from rat brain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the aryl hydrocarbon receptor nuclear translocator (Arnt1) that was completely established. Whereas ARNT2 gene expression, established by Northern blotting and in situ hybridization histochemistry, occurred selectively in brain and kidney, that of Arnt1 was ubiquitous, suggesting that the two proteins play distinct roles, presumably via dimerization and DNA binding with different partners.
Yoshiaki Fujii-kuriyama - One of the best experts on this subject based on the ideXlab platform.
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Unique and overlapping transcriptional roles of arylhydrocarbon receptor nuclear translocator (Arnt) and ARNT2 in xenobiotic and hypoxic responses.
The Journal of biological chemistry, 2006Co-Authors: Hiroki Sekine, Junsei Mimura, Masayuki Yamamoto, Yoshiaki Fujii-kuriyamaAbstract:Arnt and the homologous ARNT2 share a high degree of sequence similarity and are believed to function as obligate common partners for a number of basic helix-loop-helix (bHLH)-PAS transcription factors including arylhydrocarbon receptor (AhR) and HIFalpha. Genetic disruption of both Arnt and ARNT2 demonstrated both unique and overlapping functions in response to environmental stimuli and during mouse development. Either stably or transiently expressed Arnt/ARNT2 wild type and various mutants or chimeric constructs in Hepa1-c4 cells exhibit similar levels of hypoxic response element-driven reporter gene expression and the induction of endogenous Glut-1 through binding with HIFalpha in response to hypoxia. In contrast, we observed clear functional differences in the ability of Arnt and ARNT2 to induce xenobiotic response element-driven reporter and endogenous CYP1A1 gene expression. In contrast with Arnt, ARNT2 was practically incapable of interacting with ligand-activated AhR to induce the expression of target genes for xenobiotic-metabolizing enzymes in response to xenobiotics. The differential binding of AhR by Arnt and ARNT2 can be ascribed to a single His/Pro amino acid difference in the PASB region of Arnt and ARNT2, suggesting that the PASB/PASB interaction between bHLH-PAS transcription factors plays a selective role for their specific partner molecule.
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Defective development of secretory neurones in the hypothalamus of ARNT2‐knockout mice
Genes to cells : devoted to molecular & cellular mechanisms, 2001Co-Authors: Tomonori Hosoya, Masatsugu Ema, Masanobu Morita, Masayuki Yamamoto, Yoshihito Oda, Satoru Takahashi, Shimako Kawauchi, Yoshiaki Fujii-kuriyamaAbstract:Background Within the basic region-helix-loop-helix (bHLH)-PAS family of transcription factors, Arnt and ARNT2 play unique roles; these two factors not only heterodimerize with themselves, but also with other members of this family and they act as transcription regulators which bind to specific DNA elements. Whereas Arnt is broadly expressed in various tissues, the expression of ARNT2 is known to be limited to the neural tissues. Results To elucidate the function of ARNT2 in detail, we cloned the mouse ARNT2 gene and its gene structure was determined. We subsequently generated germ line ARNT2 mutant mice by gene targeting technology. Heterozygous ARNT2 mice were viable, but homozygous ARNT2 gene knockout mice died shortly after birth. Histological and immunological analyses revealed that the supraoptic nuclei (SON) and the paraventricular nuclei (PVN) are hypocellular. Moreover, secretory neurones identified by the expression of neurosecretory hormone such as arginine vasopressin, oxytocin, corticotrophin-releasing hormone and somatostatin are completely absent in SON and PVN in the mutant ARNT2 mice. Consistent with these observations, prospective SON and PVN neurones which express Brn2 appeared around E13.5 in the mantle zone, but no neurones which expressed the neurosecretory hormones were found in the SON and PVN regions. Conclusions These data show that the transcription factor ARNT2 controls the development of the secretory neurones at the later or final stages of differentiation rather than at the beginning stage. Strikingly similar observations have been reported with the Sim1 deficient mice. Taken together, our results demonstrate that ARNT2 is an indispensable transcription factor for the development of the hypothalamus, and suggest that ARNT2 is an obligatory partner molecule of Sim1 in the developmental process of the neuroendocrinological cell lineages.
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Transcriptionally Active Heterodimer Formation of an Arnt-like PAS Protein, Arnt3, with HIF-1a, HLF, and Clock
Biochemical and biophysical research communications, 1998Co-Authors: Sho Takahata, Kazuhiro Sogawa, Akira Kobayashi, Masatsugu Ema, Junsei Mimura, Nobuhiro Ozaki, Yoshiaki Fujii-kuriyamaAbstract:Abstract We isolated a cDNA clone encoding a polypeptide of 626 amino acids containing basic helix–loop–helix (bHLH) and PAS domains from a mouse cDNA library of P19 cells. This protein, termed Arnt3, showed the highest similarity to Arnt and ARNT2 in the bHLH and PAS regions. Arnt3 mRNA was expressed in brain, skeletal muscle, 13.5-day embryos, and P19 cells treated with retinoic acid. The partner PAS proteins of Arnt3 were searched for by the two-hybrid system in yeast, and HIF-1α, HLF, and Clock among various bHLH/PAS proteins were found. Gel mobility shift analysis using nuclear extracts from 293T cells cotransfected with Arnt3 and HIF-1α (or HLF) expression plasmids revealed that these complexes specifically bound the hypoxia-response element (HRE). Coexpression of Arnt3 and HIF-1α (or HLF) in Arnt-deficient c4 cells enhanced transcription of a reporter gene driven by the HRE sequences. We also showed that Arnt3 contained an activation domain at the C-terminal region and a repression domain between the PAS-A and PAS-B regions.
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CBP/p300 functions as a possible transcriptional coactivator of Ah receptor nuclear translocator (Arnt).
Journal of biochemistry, 1997Co-Authors: Akira Kobayashi, Kazuhiro Sogawa, Keiko Numayama-tsuruta, Yoshiaki Fujii-kuriyamaAbstract:A heterodimer of AhR (aryl hydrocarbon receptor) and Arnt (AhR nuclear translocator) conveys a transactivation signal of aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the genes for a group of drug-metabolizing enzymes. This inducible expression of the genes is inhibited by adenovirus E1A, suggesting that CBP/p300 is somehow involved in the transactivation of the genes by the AhR and Arnt heterodimer. Yeast and mammalian two hybrid systems revealed that CBP/p300 interacted with the transactivation domain of Arnt, but not with that of AhR, via the CREB-binding domain. The pull down assay using GST-Arnt hybrid protein confirmed the interaction between Arnt and CBP/p300. Considering these results and that Arnt or ARNT2 functions as a common partner in the formation of transcriptional regulators with other bHLH/PAS proteins such as AhR, HLF, and HIF-1alpha, the possibility arises that CBP/p300 is extensively involved as a coactivator in the transactivation process by bHLH/PAS (a conserved sequence motif among Per, Arnt, and Sim) heterodimer transcription factors through interaction with Arnt or ARNT2.
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cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (ARNT2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).
Molecular and cellular biology, 1996Co-Authors: K Hirose, Kazuhiro Sogawa, Masatsugu Ema, Junsei Mimura, Masanobu Morita, Hiroshi Hamada, Hideta Fujii, Yasuo Saijo, Osamu Gotoh, Yoshiaki Fujii-kuriyamaAbstract:We isolated mouse cDNA clones (ARNT2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted ARNT2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of ARNT2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. ARNT2 interacted with AhR and mouse Sim as efficiently as Arnt, and the ARNT2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of ARNT2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of ARNT2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that ARNT2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that ARNT2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, ARNT2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.