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/genéticaRESUMO
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/fisiologiaRESUMO
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 , XenobióticosRESUMO
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 fármacosRESUMO
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 ProteicaRESUMO
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 , RatosRESUMO
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/químicaRESUMO
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/metabolismoRESUMO
Polycomb group proteins are transcriptional repressors recruited to many developmental control genes. The specificity of polycomb group protein targeting is incompletely understood. Subunits of polycomb repressive complexes (PRC) are encoded by multigene families in vertebrates. Five chromodomain-containing CBX family proteins are thought to mediate chromatin association by PRC1 complexes. We visualized the recruitment of CBX proteins to chromatin using bimolecular fluorescence complementation (BiFC) analysis, wherein fragments of fluorescent proteins fused to CBX family members and histone H3 form a fluorescent complex when the CBX proteins bind to nucleosomes. Different CBX family proteins associated with nucleosomes in different subnuclear regions in both ES cells and fibroblasts. The total populations of most CBX proteins had distributions distinct from those of the chromatin-associated complexes, indicating that most of these CBX proteins were not bound to nucleosomes. The conserved chromodomain and chromobox regions of CBX proteins were dispensable for chromatin association. The absence of H3 K27 trimethylation in EED null ES cells had minimal effects on chromatin association by CBX proteins. The BiFC complexes did not colocalize with anti-trimethyl-K27 immunofluorescence, with the exception of inactive X. Metaphase spreads derived from stable cell lines with inducible CBX fusion expression revealed reciprocal patterns of chromosome association by CBX2 and CBX6 BiFC complexes. H3.2 purified from CBX2-H3.2 BiFC complexes was enriched in trimethyl-K27, dimethyl-K4, and acetyl-K9 modifications. We conclude that different CBX proteins are recruited to distinct chromatin regions through nonconserved interactions, expanding the regulatory diversity of polycomb group proteins.
Assuntos
Cromatina/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Corantes Fluorescentes , Histonas/metabolismo , Metilação , Camundongos , Nucleossomos/metabolismo , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Transporte ProteicoRESUMO
Investigations of the molecular processes that sustain life must include studies of these processes in their normal cellular environment. The bimolecular fluorescence complementation (BiFC) assay provides an approach for the visualization of protein interactions and modifications in living cells. This assay is based on the facilitated association of complementary fragments of a fluorescent protein that are fused to interaction partners. Complex formation by the interaction partners tethers the fluorescent protein fragments in proximity to each other, which can facilitate their association. The BiFC assay enables sensitive visualization of protein complexes with high spatial resolution. The temporal resolution of BiFC analysis is limited by the time required for fluorophore formation, as well as the stabilization of complexes by association of the fluorescent protein fragments. Many modifications and enhancements to the BiFC assay have been developed. The multicolor BiFC assay enables simultaneous visualization of multiple protein complexes in the same cell, and can be used to investigate competition among mutually exclusive interaction partners for complex formation in cells. The ubiquitin-mediated fluorescence complementation (UbFC) assay enables visualization of covalent ubiquitin family peptide conjugation to substrate proteins in cells. The BiFC assay can also be used to visualize protein binding to specific chromatin domains, as well as other molecular scaffolds in cells. BiFC analysis therefore provides a powerful approach for the visualization of a variety of processes that affect molecular proximity in living cells. The visualization of macromolecular interactions and modifications is of great importance owing to the central roles of proteins, nucleic acids and other macromolecular complexes in the regulation of cellular functions. This tutorial review describes the BiFC assay, and discusses the advantages and disadvantages of this experimental approach. The review will be of interest to scientists interested in the investigation of macromolecular interactions or modifications who need exquisite sensitivity for the detection of their complexes or conjugates of interest.
Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fluorescência , Microscopia de Fluorescência/métodos , Proteínas de Plantas/metabolismo , Multimerização Proteica , Proteômica , Espectrometria de Fluorescência/métodos , Espectrometria de Fluorescência/estatística & dados numéricos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Crystallin genes are selectively expressed during lens development. Maf and Sox family proteins synergistically enhanced gammaF-crystallin promoter activity in a lens cell line. Mutational analysis of the gammaF-crystallin promoter identified a composite regulatory element containing nonconsensus Maf and Sox recognition sequences. Mutations in these recognition sequences or changes in their spacing eliminated synergistic transcription activation. The transcriptional synergy was also affected by changes in the orientation of the Maf recognition sequence that had no detectable effect on binding affinity. The interaction between Maf and Sox proteins was visualized in living cells by bimolecular fluorescence complementation analysis. The N-terminal region of Maf mediated the interaction with Sox proteins in cells. Synergistic transcription activation required the N-terminal region of Maf as well as the ancillary DNA binding domain and the unique portion of the basic region that mediate specific recognition of the gammaF-crystallin promoter element. A mutation in the ancillary DNA binding domain of Maf (R288P) that has been shown to cause cataract eliminated the transcriptional activity of Maf but had no detectable effect on DNA binding in vitro. Whereas wild-type Maf was uniformly distributed in the nucleoplasm, R288P Maf was enriched in nuclear foci. Cajal bodies and gemini of coiled bodies were closely associated with the foci occupied by R288P Maf. Wild-type Maf formed complexes with Sox proteins in the nucleoplasm, whereas R288P Maf recruited Sox proteins as well as other interaction partners to the nuclear foci. The mislocalization of normal cellular proteins to these foci provides a potential explanation for the dominant disease phenotype of the R288P mutation in Maf.
Assuntos
Catarata/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Ativação Transcricional , Animais , Catarata/etiologia , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Proteínas HMGB , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/fisiologia , Humanos , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-maf , Fatores de Transcrição SOXB1 , Fatores de Transcrição , Transfecção , gama-Cristalinas/genéticaRESUMO
Many regulatory elements in eukaryotic promoters do not correspond to optimal recognition sequences for the transcription factors that regulate promoter function by binding to the elements. The sequence of the binding site may influence the structural and functional properties of regulatory protein complexes. Fos-Jun heterodimers were found to bind nonconsensus AP-1 sites in a preferred orientation. Oriented Fos-Jun heterodimer binding was attributed to nonidentical recognition of the two half-sites by Fos and Jun. Jun bound preferentially to the consensus half-site, whereas Fos was able to bind nonconsensus half-sites. The orientation of heterodimer binding affected the transcriptional cooperativity of Fos-Jun-NFAT1 complexes at composite regulatory elements in mammalian cells. Jun dimerization with Fos versus ATF2 caused it to bind opposite half-sites at nonconsensus AP-1 elements. Similarly, ATF2 bound to opposite half-sites in Fos-ATF2-NFAT1 and ATF2-Jun-NFAT1 complexes. The orientations of nonconsensus AP-1 sites within composite regulatory elements affected the cooperativity of Fos-Jun as well as Jun-Jun binding with NFAT1. Since Jun homodimers cannot bind to AP-1 sites in a preferred orientation, the effects of the orientations of nonconsensus AP-1 sites on the stabilities of Jun-Jun-NFAT1 complexes are likely to be due to asymmetric conformational changes in the two subunits of the homodimer. Nonconsensus AP-1 site orientation also affected the synergy of transcription activation between Jun homodimers and NFAT1 at composite regulatory elements. The asymmetric recognition of nonconsensus AP-1 sites can therefore influence the transcriptional activities of Fos and Jun both through effects on the orientation of heterodimer binding and through differential conformational changes in the two subunits of the dimer.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator 2 Ativador da Transcrição , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , DNA/metabolismo , Dimerização , Transferência Ressonante de Energia de Fluorescência , Luciferases/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Fatores de Transcrição NFATC , Oligonucleotídeos/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ativação TranscricionalRESUMO
Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Células COS , Divisão Celular , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-myc/genética , Frações Subcelulares/metabolismo , Células U937RESUMO
The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Células COS , Chlorocebus aethiops , Cor , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/classificação , Fatores de Ligação G-Box , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/classificação , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Transcrição/análise , Fatores de Transcrição/classificação , Dedos de ZincoRESUMO
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 , HumanosRESUMO
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/metabolismoRESUMO
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 fármacosRESUMO
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.