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1.
Immunology ; 167(1): 105-121, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35751391

RESUMO

Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1-/- deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1-/- deletions eliminated G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a-GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1-/- deletions. G9a-GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a-GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a-GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents.


Assuntos
Histona-Lisina N-Metiltransferase , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2 , Infecções por Respirovirus/metabolismo , Animais , Cromatina , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Vírus Sendai/fisiologia
2.
J Immunol ; 207(5): 1437-1447, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34400522

RESUMO

Proinflammatory cytokine gene transcription must be moderated to avoid the pathological consequences of excess cytokine production. The relationships between virus infection and the mechanisms that moderate cytokine transcription are incompletely understood. We investigated the influence of Keap1 on cytokine gene induction by Sendai virus infection in mouse embryo fibroblasts. Virus infection induced Keap1 binding to the Ifnb1, Tnf, and Il6 genes. Keap1 moderated viral induction of their transcription by mechanisms that did not require Nrf2. Keap1 was required for NF-κB p50 recruitment, but not for NF-κB p65 or IRF3 recruitment, to these genes. Keap1 formed complexes with NF-κB p50 and NF-κB p65, which were visualized using bimolecular fluorescence complementation analysis. These bimolecular fluorescence complementation complexes bound chromosomes in live cells, suggesting that Keap1 could bind chromatin in association with NF-κB proteins. Keap1 was required for viral induction of G9a-GLP lysine methyltransferase binding and H3K9me2 modification at cytokine genes. G9a-GLP inhibitors counteracted transcription repression by Keap1 and enhanced Keap1 and NF-κB recruitment to cytokine genes. The interrelationships among Keap1, NF-κB, and G9a-GLP recruitment, activities, and transcriptional effects suggest that they form a feedback circuit, which moderates viral induction of cytokine transcription. Nrf2 counteracted Keap1 binding to cytokine genes and the recruitment of NF-κB p50 and G9a-GLP by Keap1. Whereas Keap1 has been reported to influence cytokine expression indirectly through its functions in the cytoplasm, these findings provide evidence that Keap1 regulates cytokine transcription directly in the nucleus. Keap1 binds to cytokines genes upon virus infection and moderates their induction by recruiting NF-κB p50 and G9a-GLP.


Assuntos
NF-kappa B , Viroses , Animais , Citocinas , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Viroses/genética
4.
Br J Pharmacol ; 174(19): 3315-3332, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28710789

RESUMO

BACKGROUND AND PURPOSE: To further the development of new agents for the treatment of adrenocortical carcinoma (ACC), we characterized the molecular and cellular mechanisms of cytotoxicity by the adrenalytic compound ATR-101 (PD132301-02). EXPERIMENTAL APPROACH: We compared the effects of ATR-101, PD129337, and ABC transporter inhibitors on cholesterol accumulation and efflux, on cortisol secretion, on ATP levels, and on caspase activation in ACC-derived cell lines. We examined the effects of these compounds in combination with methyl-ß-cyclodextrin or exogenous cholesterol to determine the roles of altered cholesterol levels in the effects of these compounds. KEY RESULTS: ATR-101 caused cholesterol accumulation, ATP depletion, and caspase activation within 30 minutes after addition to ACC-derived cells, whereas PD129337 did not. Suppression of cholesterol accumulation by methyl-ß-cyclodextrin or exogenous cholesterol, prevented ATP depletion and caspase activation by ATR-101. ATR-101 blocked cholesterol efflux and cortisol secretion, suggesting that it inhibited ABCA1, ABCG1, and MDR1 transporters. Combinations of ABCA1, ABCG1, and MDR1 inhibitors were also cytotoxic. Combinations of ATR-101 with inhibitors of ABCG1, MDR1, or mitochondrial functions had increased cytotoxicity. Inhibitors of steroidogenesis reduced ATP depletion by ATR-101, whereas U18666A enhanced cholesterol accumulation and ATP depletion together with ATR-101. ATR-101 repressed ABCA1, ABCG1, and IDOL transcription by mechanisms that were distinct from the mechanisms that caused cholesterol accumulation. CONCLUSIONS AND IMPLICATIONS: Inhibition of multiple ABC transporters and the consequent accumulation of cholesterol mediated the cytotoxicity of ATR-101. Compounds that replicate these effects in tumours are likely to be useful in the treatment of ACC.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma Adrenocortical/metabolismo , Antineoplásicos/farmacologia , Colesterol/metabolismo , Hidrocortisona/metabolismo , Compostos de Fenilureia/farmacologia , Trifosfato de Adenosina/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Humanos
5.
Methods Enzymol ; 589: 429-455, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28336073

RESUMO

We have developed a procedure that enables visualization of the genomic loci that are bound by complexes formed by a specific combination of chromatin-binding proteins. This procedure is based on imaging bimolecular fluorescence complementation (BiFC) complexes on Drosophila polytene chromosomes. BiFC complexes are formed by the facilitated association of two fluorescent protein fragments that are fused to proteins that interact with, or are in close proximity to, each other. The intensity of BiFC complex fluorescence at individual genomic loci is greatly enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. The loci that are bound by the complexes are mapped by comparing the locations of BiFC complex fluorescence with the stereotypical banding patterns of the chromosomes. We describe strategies for the design, expression, and validation of fusion proteins for the analysis of BiFC complex binding on polytene chromosomes. We detail protocols for the preparation of polytene chromosome spreads that have been optimized for the purpose of BiFC complex visualization. Finally, we provide guidance for the interpretation of results from studies of BiFC complex binding on polytene chromosomes and for comparison of the genomic loci that are bound by BiFC complexes with those that are bound by subunits of the corresponding endogenous complexes. The visualization of BiFC complex binding on polytene chromosomes provides a unique method to visualize multiprotein complex binding at specific loci, throughout the genome, in individual cells.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/citologia , Loci Gênicos , Complexos Multiproteicos/metabolismo , Imagem Óptica/métodos , Cromossomos Politênicos/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/análise , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/análise , Cromossomos Politênicos/ultraestrutura , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo
6.
Endocr Relat Cancer ; 23(4): 1-19, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26843528

RESUMO

Adrenocortical carcinoma (ACC) generally has poor prognosis. Existing treatments provide limited benefit for most patients with locally advanced or metastatic tumors. We investigated the mechanisms for the cytotoxicity, xenograft suppression, and adrenalytic activity of ATR-101 (PD132301-02), a prospective agent for ACC treatment. Oral administration of ATR-101 inhibited the establishment and impeded the growth of ACC-derived H295R cell xenografts in mice. ATR-101 induced H295R cell apoptosis in culture and in xenografts. ATR-101 caused mitochondrial hyperpolarization, reactive oxygen release, and ATP depletion within hours after exposure, followed by cytochrome c release, caspase-3/7 activation, and membrane permeabilization. The increase in mitochondrial membrane potential occurred concurrently with the decrease in cellular ATP levels. When combined with ATR-101, lipophilic free radical scavengers suppressed the reactive oxygen release, and glycolytic precursors prevented the ATP depletion, abrogating ATR-101 cytotoxicity. ATR-101 directly inhibited F1F0-ATPase activity and suppressed ATP synthesis in mitochondrial fractions. ATR-101 administration to guinea pigs caused oxidized lipofuscin accumulation in the zona fasciculate layer of the adrenal cortex, implicating reactive oxygen release in the adrenalytic effect of ATR-101. These results support the development of ATR-101 and other adrenalytic compounds for the treatment of ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Antineoplásicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/patologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobaias , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos SCID , Mitocôndrias/fisiologia , Compostos de Fenilureia/uso terapêutico , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Carga Tumoral/efeitos dos fármacos
7.
Development ; 141(16): 3277-88, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25063457

RESUMO

Interactions among transcription factors control their physiological functions by regulating their binding specificities and transcriptional activities. We implement a strategy to visualize directly the genomic loci that are bound by multi-protein complexes in single cells in Drosophila. This method is based on bimolecular fluorescence complementation (BiFC) analysis of protein interactions on polytene chromosomes. Drosophila Keap1 (dKeap1)-CncC complexes localized to the nucleus and bound chromatin loci that were not bound preferentially by dKeap1 or CncC when they were expressed separately. dKeap1 and CncC binding at these loci was enhanced by phenobarbital, but not by tert-butylhydroquinone (tBHQ) or paraquat. Endogenous dKeap1 and CncC activated transcription of the Jheh (Jheh1, Jheh2, Jheh3) and dKeap1 genes at these loci, whereas CncC alone activated other xenobiotic response genes. Ectopic dKeap1 expression increased CncC binding at the Jheh and dKeap1 gene loci and activated their transcription, whereas dKeap1 inhibited CncC binding at other xenobiotic response gene loci and suppressed their transcription. The combinatorial chromatin-binding specificities and transcriptional activities of dKeap1-CncC complexes mediated the selective activation of different sets of genes by different xenobiotic compounds, in part through feed-forward activation of dKeap1 transcription.


Assuntos
Cromatina/química , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Cromossomos/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Corantes Fluorescentes/química , Hidroquinonas/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Dados de Sequência Molecular , Paraquat/química , Fenobarbital/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras , Glândulas Salivares/embriologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Xenobióticos
8.
Mol Cell Biol ; 34(11): 2075-91, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24687849

RESUMO

Embryonic stem (ES) cells express pluripotency-associated genes and repress differentiation-inducible genes. The activities of these genes are coordinately reversed during differentiation. The changes in the transcriptome upon conditional KAP1 knockout in ES cells overlapped with the changes during embryoid body formation. KAP1 repressed differentiation-inducible genes and derepressed pluripotency-associated genes in ES cells. KAP1 formed complexes with polycomb repressive complexes 1 (PRC1) through an interaction that was mediated by the KAP1 coiled-coil region. KAP1 and PRC1 bound cooperatively at the promoters of differentiation-inducible genes and repressed their transcription. In contrast, KAP1 bound the transcribed and flanking sequences of pluripotency-associated genes, did not enhance PRC1 binding, and derepressed their transcription. KAP1 had opposite effects on differentiation-inducible and pluripotency-associated gene transcription both in ES cells and in differentiating embryoid bodies. The region of KAP1 that mediated the interaction with PRC1 was required for KAP1 enhancement of PRC1 binding and for KAP1 repression of transcription at differentiation-inducible promoters. This region of KAP1 was not required for KAP1 suppression of PRC1 binding or for KAP1 derepression of transcription at pluripotency-associated promoters. The opposite effects of KAP1 on the transcription of differentiation-inducible versus pluripotency-associated genes contributed to the reciprocal changes in their transcription during differentiation.


Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Mapeamento Cromossômico , Células-Tronco Embrionárias/citologia , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Sequência de DNA , Transcrição Gênica/genética , Proteína 28 com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Cold Spring Harb Protoc ; 2013(10): 949-54, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24086058

RESUMO

Ubiquitin-family peptide conjugation regulates the functions and stabilities of many proteins. Numerous cellular proteins are modified by covalent conjugation of ubiquitin-family peptides to specific lysine residues. These modifications provide a flexible means for regulating the properties of the substrate proteins. Because ubiquitin can be conjugated to substrate proteins at many different sites and in many topological configurations, these modifications have the potential to confer a wide range of functional states to the modified proteins. Ubiquitin conjugation is typically detected by immunoprecipitation of a putative substrate protein followed by immunoblotting to detect ubiquitin conjugated to the substrate. However, this assay cannot be used to detect ubiquitin conjugates in live cells. It is also difficult to determine the subcellular distribution of a specific ubiquitin conjugate using this approach. To visualize ubiquitin conjugates in live cells, we have developed the ubiquitin-mediated fluorescence complementation assay, which is based on the association of fragments of fluorescent proteins when ubiquitin fused to one fragment is conjugated to a substrate protein fused to a complementary fragment. This protocol focuses on the visualization of ubiquitin conjugated in cultured mammalian cells, but it can be adapted to any cell type or aerobically grown organism that can be genetically modified to express the fusion proteins.


Assuntos
Técnicas Citológicas/métodos , Fluorometria/métodos , Coloração e Rotulagem/métodos , Ubiquitina/análise , Animais , Linhagem Celular , Humanos
10.
Cold Spring Harb Protoc ; 2013(9): 798-803, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24003202

RESUMO

Many proteins can interact with several alternative partners. The multicolor bimolecular fluorescence complementation (BiFC) assay enables simultaneous visualization of multiple protein interactions in the same cell. This assay, introduced here, is based on the fusion of fragments of fluorescent proteins that form spectrally distinct BiFC complexes to different interaction partners. These complexes can thereby be spectrally resolved and the relative amounts of complexes formed with different interaction partners can be determined. The multicolor BiFC assay enables comparison of the subcellular distributions of complexes formed with different interaction partners and allows analysis of the competition between mutually exclusive interaction partners for binding a shared partner present at limiting concentration.


Assuntos
Técnicas Citológicas/métodos , Proteínas Luminescentes/metabolismo , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Fluorescência/métodos , Coloração e Rotulagem/métodos , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
Cold Spring Harb Protoc ; 2013(9): 892-5, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24003203

RESUMO

This multicolor bimolecular fluorescence complementation procedure is designed for the analysis of alternative protein interactions in cultured mammalian cells, but it can be readily adapted to any cell type or aerobically grown organism that can be genetically modified to express the fusion proteins.


Assuntos
Técnicas Citológicas/métodos , Proteínas Luminescentes/metabolismo , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Fluorescência/métodos , Coloração e Rotulagem/métodos , Células Cultivadas , Cor , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Cold Spring Harb Protoc ; 2013(8): 714-8, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23906916

RESUMO

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. It is based on the facilitated association of two nonfluorescent fragments of a fluorescent protein fused to putative interaction partners. The intrinsic fluorescence of the active complex enables detection of protein interactions with high sensitivity, fine spatial resolution, and minimal perturbation of the cells. As discussed in more detail here, BiFC analysis requires careful consideration of the design and expression of the fusion proteins for the results to be interpretable.


Assuntos
Técnicas Citológicas/métodos , Imagem Óptica/métodos , Proteínas/metabolismo , Coloração e Rotulagem/métodos , Microscopia de Fluorescência/métodos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
Cold Spring Harb Protoc ; 2013(8): 727-31, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23906917

RESUMO

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions and modifications in living cells. It is based on the facilitated association of two nonfluorescent fragments of a fluorescent protein fused to putative interaction partners. The intrinsic fluorescence of the active complex enables detection of protein interactions with high sensitivity, fine spatial resolution, and minimal perturbation of the cells. This protocol outlines methods to analyze protein interactions in cultured mammalian cells using BiFC, but can be readily adapted to any cell type or aerobically grown organism that can be genetically modified to express the fusion proteins.


Assuntos
Técnicas Citológicas/métodos , Imagem Óptica/métodos , Proteínas/metabolismo , Coloração e Rotulagem/métodos , Animais , Células Cultivadas , Humanos , Mamíferos , Microscopia de Fluorescência/métodos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
14.
PLoS Genet ; 9(2): e1003263, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23408904

RESUMO

Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncC(K6/K6) and dKeap1(EY5/EY5) loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1(EY5/EY5) loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster , Ecdisona , Peptídeos e Proteínas de Sinalização Intracelular , Metamorfose Biológica , Fatores de Transcrição/genética , Xenobióticos/farmacologia , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ecdisona/biossíntese , Ecdisona/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Larva/genética , Larva/crescimento & desenvolvimento , Metamorfose Biológica/efeitos dos fármacos , Metamorfose Biológica/genética , Mutação , Cromossomos Politênicos/efeitos dos fármacos , Cromossomos Politênicos/genética , Proteínas Repressoras , Glândulas Salivares/citologia , Glândulas Salivares/efeitos dos fármacos , Transdução de Sinais/genética , Ativação Transcricional/efeitos dos fármacos
15.
J Biol Chem ; 287(38): 31833-44, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22843696

RESUMO

ATF2-Jun, IRF3, and HMGI recognize a composite regulatory element within the interferon-ß enhancer (IFNb). Cooperative ATF2-Jun-IRF3 complex formation at IFNb has been proposed to require a fixed orientation of ATF2-Jun binding. Our results show that ATF2-Jun heterodimers bound IFNb in both orientations alone and in association with IRF3 and HMGI. Two sets of symmetrically located amino acid residues in ATF2 and Jun facilitated the interactions between heterodimers bound in opposite orientations and IRF3 at IFNb. IRF3 and HMGI bound IFNb in association with both orientations of ATF2-Jun heterodimers with the same cooperativity. ATF2-Jun heterodimers that bound IFNb in opposite orientations in vitro had different effects on interferon-ß gene transcription when they were co-expressed with IRF3 in cultured cells. These heterodimers had different transcriptional activities at different endogenous genes. Different regions of ATF2 and Jun mediated their orientation-dependent transcriptional activities at different genes. These studies revealed that cooperative DNA binding does not require a unique nucleoprotein complex configuration, and that transcription factor complexes that bind the same enhancer in different configurations can have different transcriptional activities.


Assuntos
DNA/química , Interferon beta/química , Fatores de Transcrição/química , Transcrição Gênica , Fator 2 Ativador da Transcrição/química , Dimerização , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Proteína HMGA1a/química , Humanos , Fator Regulador 3 de Interferon/química , Proteínas Quinases JNK Ativadas por Mitógeno/química , Nucleoproteínas/química , Oligonucleotídeos/química , Ligação Proteica , Conformação Proteica
16.
J Neurosci ; 31(37): 13224-35, 2011 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-21917805

RESUMO

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.


Assuntos
Transporte Axonal/fisiologia , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Potenciais da Membrana , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos
17.
Mol Cell Biol ; 31(14): 3068-80, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21606195

RESUMO

Cardiac homeostasis is maintained by a balance of growth-promoting and growth-modulating factors. Sustained elevation of calcium signaling can induce cardiac hypertrophy through activation of Nfat family transcription factors. FoxP family transcription factors are known to interact with Nfat proteins and to modulate their transcriptional activities in lymphocytes. We investigated FoxP1 interaction with Nfat3 (Nfatc4) and their effects on transcription of hypertrophy-associated genes in neonatal rat cardiomyocytes. FoxP1-Nfat3 complexes were visualized using bimolecular fluorescence complementation (BiFC) analysis. Calcineurin activation induced FoxP1-Nfat3 BiFC complex formation. Amino acid substitutions in the predicted interaction interface inhibited it. FoxP1 repressed hypertrophy-associated genes (Myh7, Rcan1, Cx43, Anf, and Bnp) and counteracted their activation by constitutively nuclear Nfat3 (cnNfat3). In contrast, FoxP1 activated genes that maintain normal heart functions (Myh6 and p57Kip2) and cnNfat3 counteracted their activation by FoxP1. Amino acid substitutions in FoxP1 or cnNfat3 that inhibited their interaction abrogated the activation of hypertrophy-associated gene transcription by cnNfat3 and the repression of these genes by FoxP1. FoxP1 and Nfat3 co-occupied the promoter regions of hypertrophy-associated genes in neonatal and adult heart tissue. FoxP1 counteracted hypertrophic cardiomyocyte growth, and connexin 43 mislocalization caused by cnNfat3 expression. These data suggest that the opposing transcriptional activities of FoxP1 and Nfat3 maintain cardiomyocyte homeostasis.


Assuntos
Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Fatores de Transcrição Forkhead/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Fatores de Transcrição NFATC/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/fisiologia , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética
18.
Mol Cell Biol ; 31(10): 2100-10, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21402785

RESUMO

Polycomb group (PcG) proteins control the epigenetic inheritance of transcription regulatory states during development. Progression from pluripotency to differentiation requires the concurrent activation and repression of different PcG target genes. We found that REST and nine REST-associated proteins copurified with Cbx family PcG proteins from mouse embryonic stem (ES) cells. REST interacted with Cbx proteins in live cells and coprecipitated with endogenous Ring1b. Endogenous PRC1 subunits occupied all sites tested that were bound by REST in ES cells. Antibodies directed against different PRC1 subunits precipitated proximal versus distal RE1 elements with opposite relative efficiencies, suggesting that PRC1 bound different sites in distinct configurations. Deletion of the amino-terminal region of REST (Rest(ΔN) knockout) as well as short hairpin RNA depletion of REST (REST knockdown) in ES cells reduced PRC1 binding at distal RE1 elements and increased PRC1 binding at proximal RE1 elements. Rest(ΔN) and PRC1 subunit knockout as well as REST and PRC1 subunit knockdown had similar relative effects on transcription of neuronal genes in ES cells, derepressing genes with distal, but not genes with proximal, RE1 elements. In differentiating neurons, Rest(ΔN) knockout reduced PRC1 occupancy and derepressed transcription at distal RE1 elements but increased PRC1 occupancy and repressed transcription at proximal RE1 elements. The opposite effects of REST on PRC1 occupancy at different RE1 elements contributed to the gene-specific control of PRC1 functions during ES cell differentiation.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Repressoras/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Camundongos , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , RNA Interferente Pequeno , Proteínas Repressoras/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases
19.
Exp Cell Res ; 317(6): 706-23, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21122806

RESUMO

How the co-ordinated events of gene activation and silencing during cellular differentiation are influenced by spatial organization of the cell nucleus is still poorly understood. Little is known about the molecular mechanisms controlling subnuclear distribution of transcription factors, and their interplay with nuclear proteins that shape chromatin structure. Here we show that C/EBPß not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBPß dimers localize in different nuclear domains. Using BiFC in living cells, we show that LAP (Liver Activating Protein) homodimers localize in euchromatin and heterochromatin. In contrast, LIP (Liver Inhibitory Protein) homodimers localize exclusively in heterochromatin. Importantly, their differential subnuclear distribution mirrors the site for interaction with HP1α. HP1α inhibits LAP transcriptional capacity and occupies the promoter of the C/EBPß-dependent gene c/ebpα in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1α binding decreases from c/ebpα promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBPß associated with chromatin or nucleoskeleton, and dynamic changes in their interaction with HP1α, play key roles in the regulation of C/EBP target genes during adipogenesis.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Células 3T3 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular , Núcleo Celular/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Dimerização , Regulação da Expressão Gênica , Humanos , Camundongos , Microscopia de Fluorescência , Proteínas Nucleares/química
20.
Biol Cell ; 102(9): 525-37, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20590528

RESUMO

BACKGROUND INFORMATION: Cell fusion is known to underlie key developmental processes in humans and is postulated to contribute to tissue maintenance and even carcinogenesis. The mechanistic details of cell fusion, especially between different cell types, have been difficult to characterize because of the dynamic nature of the process and inadequate means to track fusion products over time. Here we introduce an inducible system for detecting and tracking live cell fusion products in vitro and potentially in vivo. This system is based on BiFC (bimolecular fluorescence complementation) analysis. In this approach, two proteins that can interact with each other are joined to fragments of a fluorescent protein and are expressed in separate cells. The interaction of said proteins after cell fusion produces a fluorescent signal, enabling the identification and tracking of fusion products over time. RESULTS: Long-term tracking of fused p53-deficient cells revealed that hybrid cells were capable of proliferation. In some cases, proliferation was preceded by nuclear fusion and division was asymmetric (69%+/-2% of proliferating hybrids), suggesting chromosomal instability. In addition, asymmetric division following proliferation could give rise to progeny indistinguishable from unfused counterparts. CONCLUSIONS: These results support the possibility that the chromosomal instability characteristic of tumour cells may be incurred as a consequence of cell fusion and suggest that the role of cell fusion in carcinogenesis may have been masked to this point for lack of an inducible method to track cell fusion. In sum, the BiFC-based approach described here allows for comprehensive studies of the mechanism and biological impact of cell fusion in nature.


Assuntos
Fusão Celular , Instabilidade Cromossômica , Células Híbridas/fisiologia , Medições Luminescentes/métodos , Microscopia de Fluorescência/métodos , Animais , Células COS , Divisão Celular , Proliferação de Células , Chlorocebus aethiops , Fluorescência , Células Híbridas/citologia , Proteínas Luminescentes/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo
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