RESUMO
Activation of adhesion receptor GPR110 by the endogenous ligand synaptamide promotes neurogenesis, neurite growth, and synaptogenesis in developing brains through cAMP signal transduction. However, interacting partners of GPR110 and their involvement in cellular function remain unclear. Here, we demonstrate using chemical crosslinking, affinity purification, and quantitative mass spectrometry that GPR110 interacts with the tight junction adhesion protein occludin. By removing non-specific partners by comparing the binding proteins of GPR110 WT and an inactive mutant exhibiting impaired surface expression, occludin was distinguished as a true binding partner which was further confirmed by reciprocal co-immunoprecipitation assay. Deletion of GPR110 in mice led to the disruption of blood-brain barrier (BBB) and reduced occludin phosphorylation at Y285 in the brain. The Y285 phosphorylation increased upon the ligand-induced activation of GPR110. These data suggest an important role of GPR110-occludin interaction in BBB function and association of previously unknown GPR110-dependent occludin phosphorylation at Y285 with BBB integrity.
RESUMO
It is extremely difficult to achieve functional recovery after axonal injury in the adult central nervous system. The activation of G-protein coupled receptor 110 (GPR110, ADGRF1) has been shown to stimulate neurite extension in developing neurons and after axonal injury in adult mice. Here, we demonstrate that GPR110 activation partially restores visual function impaired by optic nerve injury in adult mice. Intravitreal injection of GPR110 ligands, synaptamide and its stable analogue dimethylsynaptamide (A8) after optic nerve crush significantly reduced axonal degeneration and improved axonal integrity and visual function in wild-type but not gpr110 knockout mice. The retina obtained from the injured mice treated with GPR110 ligands also showed a significant reduction in the crush-induced loss of retinal ganglion cells. Our data suggest that targeting GPR110 may be a viable strategy for functional recovery after optic nerve injury.
Assuntos
Traumatismos do Nervo Óptico , Animais , Camundongos , Axônios , Ligantes , Camundongos Knockout , Compressão Nervosa , Regeneração Nervosa/fisiologia , Receptores Acoplados a Proteínas G/genética , Retina , Células Ganglionares da Retina/fisiologiaRESUMO
Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.
Assuntos
Etanolaminas/farmacologia , Proteínas Oncogênicas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Sítios de Ligação , Etanolaminas/metabolismo , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.
Assuntos
Tecido Adiposo/citologia , Fosfatase Alcalina/metabolismo , Humor Aquoso/fisiologia , Temperatura Alta , Células-Tronco Mesenquimais/enzimologia , Osteogênese/fisiologia , Análise de Variância , Humor Aquoso/química , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Proteínas do Citoesqueleto/farmacologia , Relação Dose-Resposta a Droga , Proteínas do Olho/farmacologia , Glicoproteínas/farmacologia , HumanosRESUMO
Myocilin is a secreted glycoprotein that belongs to a family of olfactomedin domain-containing proteins. Although myocilin is detected in several ocular and nonocular tissues, the only reported human pathology related to mutations in the MYOCILIN gene is primary open-angle glaucoma. Functions of myocilin are poorly understood. Here we demonstrate that myocilin is a mediator of oligodendrocyte differentiation and is involved in the myelination of the optic nerve in mice. Myocilin is expressed and secreted by optic nerve astrocytes. Differentiation of optic nerve oligodendrocytes is delayed in Myocilin-null mice. Optic nerves of Myocilin-null mice contain reduced levels of several myelin-associated proteins including myelin basic protein, myelin proteolipid protein, and 2'3'-cyclic nucleotide 3'-phosphodiesterase compared with those of wild-type littermates. This leads to reduced myelin sheath thickness of optic nerve axons in Myocilin-null mice compared with wild-type littermates, and this difference is more pronounced at early postnatal stages compared with adult mice. Myocilin also affects differentiation of oligodendrocyte precursors in vitro. Its addition to primary cultures of differentiating oligodendrocyte precursors increases levels of tested markers of oligodendrocyte differentiation and stimulates elongation of oligodendrocyte processes. Myocilin stimulation of oligodendrocyte differentiation occurs through the NgR1/Lingo-1 receptor complex. Myocilin physically interacts with Lingo-1 and may be considered as a Lingo-1 ligand. Myocilin-induced elongation of oligodendrocyte processes may be mediated by activation of FYN and suppression of RhoA GTPase.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/fisiologia , Nervo Óptico/citologia , Receptores de Superfície Celular/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Espinais/citologia , Regulação da Expressão Gênica/genética , Glicoproteínas/genética , Humanos , Técnicas In Vitro , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas da Mielina/genética , Proteínas do Tecido Nervoso/genética , Receptor Nogo 1 , Oligodendroglia/ultraestrutura , Receptores de Superfície Celular/genética , Células-Tronco/fisiologiaRESUMO
Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.
Assuntos
Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/metabolismo , Animais , Apoptose/fisiologia , Caspase 7/genética , Caspase 7/metabolismo , Sobrevivência Celular/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glicoproteínas/genética , Células HEK293 , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologiaRESUMO
The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Bainha de Mielina/metabolismo , Sistema Nervoso Periférico/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Glaucoma/metabolismo , Glicoproteínas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Mutação , Bainha de Mielina/genética , Fosforilação , Nós Neurofibrosos/metabolismo , Nervo Isquiático/metabolismo , Transdução de SinaisRESUMO
Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse, rat, and human bone marrow, with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage, which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs, which was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally, cortical bone thickness and trabecular volume, as well as the expression level of osteopontin, a known factor of bone remodeling and osteoblast differentiation, were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for myocilin in bone formation and/or maintenance in vivo.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas do Citoesqueleto/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas do Olho/genética , Glicoproteínas/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Mutantes , Osteoblastos/citologia , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/fisiologiaRESUMO
The MYOCILIN gene encodes a secreted glycoprotein which is highly expressed in eye drainage structures. Mutations in this gene may lead to juvenile open-angle glaucoma and adult onset primary open-angle glaucoma, one of the leading causes of irreversible blindness in the world. Functions of wild-type myocilin are still unclear. We have recently demonstrated that myocilin is a modulator of Wnt signaling and may affect actin cytoskeleton organization. Here we report that myocilin and its naturally occurring proteolytic fragments, similar to Wnt3a, are able to stimulate trabecular meshwork, NIH3T3, and FHL124 cell migration with the N-terminal proteolytic fragment of myocilin lacking the olfactomedin domain producing the highest stimulatory effect. Stimulation of cell migration occurs through activation of the integrin-focal adhesion kinase (FAK)-serine/threonine kinase (AKT) signaling pathway. Inhibition of FAK by siRNA reduced the stimulatory action of myocilin by threefold. Activation of several components of this signaling pathway was also demonstrated in the eyes of transgenic mice expressing elevated levels of myocilin in the eye drainage structures. These data extend the similarities between actions of myocilin and Wnt proteins acting through a ß-catenin-independent mechanism. The modification of the migratory ability of cells by myocilin may play a role in normal functioning of the eye anterior segment and its pathology including glaucoma.
Assuntos
Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glicoproteínas/metabolismo , Integrinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Malha Trabecular/enzimologia , Animais , Movimento Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Ativação Enzimática , Proteínas do Olho/genética , Quinase 1 de Adesão Focal/genética , Glicoproteínas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Malha Trabecular/efeitos dos fármacos , Transfecção , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
It is well documented that mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. However, the functions of wild-type myocilin are still not well understood. To study the functions of human myocilin and its two proteolytic fragments, these proteins were expressed in HEK293 cells. Conditioned medium from myocilin-expressing cells, as well as purified myocilin, induced the formation of stress fibers in primary cultures of human trabecular meshwork or NIH 3T3 cells. Stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin, as well as secreted inhibitors of Wnt signaling, secreted Frizzled-related protein 1 (sFRP1) or sFRP3, and beta-catenin small interfering RNA. Interaction of myocilin with sFRP1, sFRP3, and several Frizzled receptors was confirmed by immunoprecipitation experiments and by binding of myocilin to the surface of cells expressing cysteine-rich domains of different Frizzled and sFRPs. Treatment of NIH 3T3 cells with myocilin and its fragments induced intracellular redistribution of beta-catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of beta-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of beta-catenin in these tissues. Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, since myocilin modulates Wnt signaling by interacting with components of this signaling pathway.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas do Olho/fisiologia , Glicoproteínas/fisiologia , Proteínas Wnt/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Glicoproteínas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , beta Catenina/metabolismoRESUMO
Protein transduction (PT) is a method for delivering proteins into mammalian cells. PT is accomplished by linking a small peptide tag--called a PT domain (PTD)--to a protein of interest, which generates a functional fusion protein that can penetrate efficiently into mammalian cells. In order to study the functions of a transcription factor (TF) of interest, expression plasmids that encode the TF often are transfected into mammalian cells. However, the efficiency of DNA transfection is highly variable among different cell types and is usually very low in primary cells, stem cells and tumor cells. Zinc-finger transcription factors (ZF-TFs) can be tailor-made to target almost any gene in the human genome. However, the extremely low efficiency of DNA transfection into cancer cells, both in vivo and in vitro, limits the utility of ZF-TFs. Here, we report on an artificial ZF-TF that has been fused to a well-characterized PTD from the human immunodeficiency virus-1 (HIV-1) transcriptional activator protein, Tat. This ZF-TF targeted the endogenous promoter of the human VEGF-A gene. The PTD-attached ZF-TF was delivered efficiently into human cells in vitro. In addition, the VEGF-A-specific transcriptional repressor retarded the growth rate of tumor cells in a mouse xenograft experiment.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição/genética , Dedos de Zinco , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Nus , Neoplasias/patologia , Neoplasias/terapia , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Gene expression is regulated at the transcriptional and post-transcriptional levels. Therefore, in order to achieve a high level of silencing, which includes minimizing any residual expression of a target gene, suppression at both the transcriptional and post-transcriptional levels is required. In this study, we describe a new method for highly efficient gene silencing that combines zinc finger protein-mediated transcriptional repression and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events. To measure the amount of gene expression under various conditions, we used a luciferase reporter gene that was driven by a variety of promoters, including that of the human vascular endothelial growth factor-A (VEGF-A) gene. We also measured expression of the endogenous VEGF-A gene. Inhibition of gene expression by each of the two individual technologies was effective, but in-depth analyses revealed residual expression of the target gene. The combination of specific zinc finger transcription factors and siRNAs greatly enhanced the silencing of the human VEGF-A gene, not only when cells were grown in the presence of normal amounts of oxygen but also under conditions of hypoxic stimulation. These results suggest that a bi-level approach to the silencing of VEGF-A expression may be clinically beneficial as part of a cancer treatment protocol.
Assuntos
Inativação Gênica , Interferência de RNA , Fator A de Crescimento do Endotélio Vascular/genética , Hipóxia Celular , Linhagem Celular , Genes Reporter , Humanos , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Dedos de ZincoRESUMO
Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.
Assuntos
HIV-1/metabolismo , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Sítios de Ligação , Linhagem Celular , Sistema Livre de Células , Cloranfenicol O-Acetiltransferase/metabolismo , Ciclina T , Ciclinas/química , Deleção de Genes , Produtos do Gene tat/metabolismo , HIV , Células HeLa , Humanos , Modelos Biológicos , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Polimerase II/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Zinco/química , Dedos de Zinco , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.