RESUMO
Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype or, indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity and thus motor coordination.
Assuntos
Padronização Corporal , Neurônios Motores/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Animais , Eletromiografia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Membro Posterior/inervação , Camundongos , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
The precision with which motor neurons innervate target muscles depends on a regulatory network of Hox transcription factors that translates neuronal identity into patterns of connectivity. We show that a single transcription factor, FoxP1, coordinates motor neuron subtype identity and connectivity through its activity as a Hox accessory factor. FoxP1 is expressed in Hox-sensitive motor columns and acts as a dose-dependent determinant of columnar fate. Inactivation of Foxp1 abolishes the output of the motor neuron Hox network, reverting the spinal motor system to an ancestral state. The loss of FoxP1 also changes the pattern of motor neuron connectivity, and in the limb motor axons appear to select their trajectories and muscle targets at random. Our findings show that FoxP1 is a crucial determinant of motor neuron diversification and connectivity, and clarify how this Hox regulatory network controls the formation of a topographic neural map.
Assuntos
Diferenciação Celular , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurônios Motores/metabolismo , Proteínas Repressoras/metabolismo , Medula Espinal/metabolismo , Animais , Embrião de Galinha , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios Motores/citologia , Proteínas Repressoras/genética , Medula Espinal/citologiaRESUMO
The molecular pathways regulating cell lineage determination and regeneration in epithelial tissues are poorly understood. The secretory epithelium of the lung is required for production of mucus to help protect the lung against environmental insults, including pathogens and pollution, that can lead to debilitating diseases such as asthma and chronic obstructive pulmonary disease. We show that the transcription factors Foxp1 and Foxp4 act cooperatively to regulate lung secretory epithelial cell fate and regeneration by directly restricting the goblet cell lineage program. Loss of Foxp1/4 in the developing lung and in postnatal secretory epithelium leads to ectopic activation of the goblet cell fate program, in part, through de-repression of the protein disulfide isomerase anterior gradient 2 (Agr2). Forced expression of Agr2 is sufficient to promote the goblet cell fate in the developing airway epithelium. Finally, in a model of lung secretory cell injury and regeneration, we show that loss of Foxp1/4 leads to catastrophic loss of airway epithelial regeneration due to default differentiation of secretory cells into the goblet cell lineage. These data demonstrate the importance of Foxp1/4 in restricting cell fate choices during development and regeneration, thereby providing the proper balance of functional epithelial lineages in the lung.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Pulmão/metabolismo , Mucoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Southern Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina , Fatores de Transcrição Forkhead/genética , Células Caliciformes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mucoproteínas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas , Reação em Cadeia da Polimerase , Regeneração/fisiologia , Proteínas Repressoras/genéticaRESUMO
The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian beta-globin loci have served as a model for gene regulation during development. Transgenic mice containing the human beta-globin locus, consisting of the linked embryonic (epsilon), fetal (gamma) and adult (beta) genes, have been used as a system to investigate the temporal switch from fetal to adult haemoglobin, as occurs in humans. Here we show that the human gamma-globin (HBG) genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human gamma-globin expression identified by genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human gamma-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of beta-globin gene regulation in mice and humans, we have shown that alterations in the expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Embrião de Mamíferos/metabolismo , Evolução Molecular , Feto/metabolismo , Inativação Gênica , Hematopoese , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Repressoras , Especificidade da Espécie , Globinas beta/genética , gama-Globinas/genéticaRESUMO
Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.
Assuntos
Microdomínios da Membrana/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Antígenos/metabolismo , Linfócitos B/enzimologia , Proteínas de Ligação a DNA , Humanos , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Lipoilação , Ativação Linfocitária , Microdomínios da Membrana/enzimologia , Camundongos , Mutação/genética , Oncogenes , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Tirosina Quinases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição TFII/metabolismo , Transcrição GênicaRESUMO
The presence of actin in the nucleus has been well established, and several studies have implicated nuclear actin in transcriptional regulation. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a member of the WASP family of proteins; these proteins function in the cytoplasm as key regulators of cortical actin filament. Interestingly, N-WASP has also been observed in the nucleus. However, a potential nuclear function for N-WASP has not been established. Here, we report the identification of nuclear N-WASP within a large nuclear-protein complex containing PSF-NonO (polypyrimidine-tract-binding-protein-associated splicing factor-non-Pou-domain octamer-binding protein/p54(nrb)), nuclear actin and RNA polymerase II. The PSF-NonO complex is involved in the regulation of many cellular processes, such as transcription, RNA processing, DNA unwinding and repair. We demonstrate that the interaction of N-WASP with the PSF-NonO complex can couple N-WASP with RNA polymerase II to regulate transcription. We also provide evidence that the potential function of N-WASP in promoting polymerization of nuclear actins has an important role in this process. Based on these results, we propose a nuclear function for N-WASP in transcriptional regulation.
Assuntos
Actinas/biossíntese , Núcleo Celular/metabolismo , RNA Polimerase II/metabolismo , Elementos Reguladores de Transcrição/genética , Transcrição Gênica/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Núcleo Celular/genética , Proteínas de Ligação a DNA , Humanos , Substâncias Macromoleculares/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB , Polímeros/metabolismo , RNA Polimerase II/genética , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcgamma receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcgammaRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcgammaRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcgammaRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcgammaRI but not to the other activating receptors (FcgammaRIIa, FcgammaRIIIa) nor to the inhibitory receptor, FcgammaRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fcgamma receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcgammaR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.
Assuntos
Células Dendríticas/imunologia , Imunoglobulina G/genética , Monócitos/imunologia , Receptores de IgG/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Escherichia coli/imunologia , Citometria de Fluxo , Engenharia Genética , Variação Genética , Glicosilação , Humanos , Modelos Moleculares , Conformação Proteica , Receptores de IgG/química , Proteínas Recombinantes/imunologia , TrastuzumabRESUMO
Proper thymocyte development is required to establish T-cell central tolerance and to generate naive T cells, both of which are essential for T-cell homeostasis and a functional immune system. Here we demonstrate that the loss of transcription factor Foxp1 results in the abnormal development of T cells. Instead of generating naive T cells, Foxp1-deficient single-positive thymocytes acquire an activated phenotype prematurely in the thymus and lead to the generation of peripheral CD4(+) T and CD8(+) T cells that exhibit an activated phenotype and increased apoptosis and readily produce cytokines upon T-cell receptor engagement. These results identify Foxp1 as an essential transcriptional regulator for thymocyte development and the generation of quiescent naive T cells.
Assuntos
Diferenciação Celular/genética , Fatores de Transcrição Forkhead/fisiologia , Proteínas Repressoras/fisiologia , Linfócitos T/fisiologia , Timo/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Proteínas Repressoras/genética , Linfócitos T/metabolismo , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
To determine the underlying mechanism of Foxp1/2/4-mediated transcriptional repression, a yeast two-hybrid screen was performed that identified p66beta, a transcriptional repressor and component of the NuRD chromatin-remodeling complex. We show that direct interactions between Foxp1/4 and p66beta are mediated by the CR2 domain within p66beta and the zinc finger/leucine zipper repression domain found in Foxp1/2/4. These direct interactions are functionally relevant as overexpression of p66beta in combination with Foxp factors cooperatively represses Foxp target gene expression, whereas loss of p66 and Foxp factors results in de-repression of endogenous Foxp target genes in lung epithelial cells. Moreover, the NuRD components HDAC1/2 associate in a macromolecular complex with Foxp proteins, and loss of expression or inhibition of HDAC1/2 activity leads to de-repression of Foxp target gene expression. Importantly, we show in vivo that Foxp1 and HDAC2 act cooperatively to regulate expression of the cytoprotective cytokine interleukin-6, which results in increased resistance to hyperoxic lung injury in Foxp1/HDAC2 compound mutant animals. These data reveal an important interaction between the Foxp transcription factors and the NuRD chromatin-remodeling complex that modulates transcriptional repression critical for the lung epithelial injury response.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Hiperóxia/metabolismo , Interleucina-6/biossíntese , Pulmão/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteínas Repressoras/metabolismo , Mucosa Respiratória/lesões , Mucosa Respiratória/metabolismo , Animais , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Hiperóxia/genética , Interleucina-6/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Camundongos Mutantes , Células NIH 3T3 , Proteínas Repressoras/genética , Técnicas do Sistema de Duplo-Híbrido , Dedos de ZincoRESUMO
B-cell regulator of immunoglobulin heavy chain transcription (Bright)/ARID3a, an A+T-rich interaction domain protein, was originally discovered in B lymphocyte lineage cells. However, expression patterns and high lethality levels in knockout mice suggested that it had additional functions. Three independent lines of evidence show that functional inhibition of Bright results in increased developmental plasticity. Bright-deficient cells from two mouse models expressed a number of pluripotency-associated gene products, expanded indefinitely, and spontaneously differentiated into cells of multiple lineages. Furthermore, direct knockdown of human Bright resulted in colonies capable of expressing multiple lineage markers. These data suggest that repression of this single molecule confers adult somatic cells with new developmental options.
Assuntos
Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/deficiência , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Dominantes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Interferência de RNA , Teratoma/genética , Teratoma/metabolismo , Fatores de Transcrição/genéticaRESUMO
Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunoglobulinas/genética , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Meios de Cultura Livres de Soro/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Ácidos Graxos Insaturados/farmacologia , Regulação da Expressão Gênica , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Regiões de Interação com a Matriz , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Sinais de Localização Nuclear , Oncogenes/efeitos dos fármacos , Oncogenes/genética , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fase S/genética , Transativadores/efeitos dos fármacos , Transativadores/genética , Fatores de Transcrição , Células Tumorais Cultivadas , Proteína Exportina 1RESUMO
Intrathymic signals induce the differentiation of immature CD4(+)CD8(+) double positive (DP) thymocytes into mature CD4(+) or CD8(+) single positive (SP) T cells. The transcriptional mechanism by which CD8 lineage is determined is not fully understood. The best evidence, which favors the kinetic signaling/coreceptor reversal model, indicates that signaled DP thymocytes terminate CD8 transcription prior to their subsequent re-initiation of CD8 transcription and ultimate differentiation into CD8SP T cells. We and others have shown that CD8 lineage commitment is severely perturbed in mice in which expression of the transcription factor SATB1 is either conventionally knocked out or T cell-specifically knocked down. Here, we demonstrate that, as with normal thymocytes, cultured SATB1-deficient DP thymocytes inactivate CD8 coreceptor transcription following receipt of signals (PMA plus ionomycin) that mimic TCR-mediated positive selection. However, this terminated CD8 transcription is not re-initiated by signals (IL-7) conducive to CD8 differentiation in SATB1-deficient DP. We show that SATB1 specifically binds to a cis-regulatory element within the CD8 enhancer (E8(III)) known to be required for coreceptor reversal. A requirement in CD8 coreceptor reversal identifies SATB1 as an essential trans-regulator of CD8 lineage fate, whose action may be mediated via recruitment to the E8(III) DP enhancer.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação à Região de Interação com a Matriz/imunologia , Receptores de Superfície Celular/imunologia , Animais , Sítios de Ligação , Linfócitos T CD8-Positivos/efeitos dos fármacos , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-7/farmacologia , Proteínas de Ligação à Região de Interação com a Matriz/deficiência , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Ligação Proteica/efeitos dos fármacosRESUMO
The direct correlation between levels of heat shock protein expression and efficiency of its tissue protection function motivates this study of how thermal doses can be used for an optimal stress protocol design. Heat shock protein 70 (HSP70) expression kinetics were visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a microscope heating stage using green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP(p)-HSP70-GFP which expresses an HSP70-GFP fusion protein under control of the HSP70 promoter. Expression levels were validated by western blot analysis. Transfected cells were heated on a controlled temperature microscope stage at 42 degrees C for a defined period, then shifted to 37 degrees C for varied post-heating times. The expression of HSP70-GFP and its sub-cellular localization were visualized via fluorescence microscopy. The progressive expression kinetics were measured by quantitative analysis of serial fluorescence images captured during heating protocols from 1 to 2 h and post-heating times from 0 to 20 h. The results show two sequential peaks in HSP70 expression at approximately 3 and 12 h post-heat shock. A progressive translocation of HSP70 from the cytoplasm to the nucleus was observed from 6 to 16 h. We conclude that we have successfully combined molecular cloning and optical imaging to study HSP70 expression kinetics. The kinetic profile for HSP70-GFP fusion protein is consistent with the endogenous HSP70. Furthermore, information on dynamic intracellular translocation of HSP70 was extracted from the same experimental data.
Assuntos
Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Microscopia de Fluorescência/métodos , Frações Subcelulares/metabolismo , Animais , Bovinos , Células Cultivadas , Células Endoteliais/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Calefação/métodos , Cinética , Taxa de Depuração Metabólica , Perfusão , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/ultraestrutura , TemperaturaRESUMO
PSF (PTB-associated splicing factor) is a multi-functional protein that participates in transcription and RNA processing. While phosphorylation of PSF has been shown to be important for some functions, the sites and the kinases involved are not well understood. Although PSF does not contain a typical RS domain, we report here that PSF is phosphorylated in vivo to generate an epitope(s) that can be recognized by a monoclonal antibody specific for phosphorylated RS motifs within SR proteins. PSF can be phosphorylated by human and yeast SR kinases in vivo and in vitro at an isolated RS motif within its N terminus. A functional consequence of SR phosphorylation of PSF is to inhibit its binding to the 3' polypyrimidine tract of pre-mRNA. These results indicate that PSF is a substrate of SR kinases whose phosphorylation regulates its RNA binding capacity and ultimate biological function.
Assuntos
Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Fosfo-Específicos/imunologia , Humanos , Dados de Sequência Molecular , Fosforilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/imunologia , Pirimidinas/metabolismo , Especificidade por SubstratoRESUMO
Uncensored exchange of scientific results hastens progress. Open Access does not stop at the removal of price and permission barriers; still, censorship and reading disabilities, to name a few, hamper access to information. Here, we invite the scientific community and the public to discuss new methods to distribute, store and manage literature in order to achieve unfettered access to literature.
Assuntos
Acesso à Informação , Internet , Bases de Dados Bibliográficas , Humanos , Publicações Periódicas como Assunto/tendências , PubMed , Editoração/tendênciasRESUMO
BACKGROUND: Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2. RESULTS: Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation. CONCLUSION: We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.
Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/química , Metiltransferases/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina/química , Histona-Lisina N-Metiltransferase/química , Humanos , Lisina/química , Metiltransferases/biossíntese , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Homologia de Sequência de Aminoácidos , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
BACKGROUND: Chromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development. RESULTS: Alternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles. CONCLUSION: We propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.
Assuntos
Proteínas de Transporte/metabolismo , Centro Germinativo/metabolismo , Linfoma de Células B/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Processamento Alternativo/genética , Animais , Western Blotting , Células COS , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Perfilação da Expressão Gênica , Centro Germinativo/patologia , Células HeLa , Humanos , Imunoprecipitação , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/análise , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas RepressorasRESUMO
The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.
Assuntos
Córtex Suprarrenal/enzimologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/genética , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Western Blotting , Núcleo Celular/genética , Células Cultivadas , Cicloeximida/farmacologia , Fatores de Transcrição Fushi Tarazu , Regulação Enzimológica da Expressão Gênica/genética , Histona Desacetilases/genética , Proteínas de Homeodomínio , Humanos , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Inibidores da Síntese de Proteínas/farmacologia , Receptores Citoplasmáticos e Nucleares , Fatores de Processamento de Serina-Arginina , Fator Esteroidogênico 1 , Transcrição Gênica/genéticaRESUMO
Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a(lox/lox) deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/metabolismo , Ativação Transcricional , Recombinação V(D)J , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Células Precursoras de Linfócitos B/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras , Regulação para CimaRESUMO
In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.