RESUMO
Transcriptional up-regulation of the plasminogen activator inhibitor type-2 (PAI-2) gene is a major response to cellular stress. The expression of PAI-2 is induced by a variety of cytokines and growth factors that act in a cell type- and differentiation stage-dependent manner. We previously reported that the human SERPINB2 gene promoter is controlled by three major transcription regulatory domains: an inducible proximal promoter, an upstream silencer (PAUSE-1), and a distal transactivator region between -5100 and -3300, which appears to overcome inhibition mediated by the silencer. The distal transactivator region is inducible by the phorbol ester PMA, a potent activator of the protein kinase C (PKC) pathway that is a powerful inducer of PAI-2 gene expression in monocytes, macrophages, and myelomonocytic cells as well as in epidermal keratinocytes. Here we show that a 21-bp region (-4952/-4932), containing an AP-1 element, is both necessary and sufficient for PMA-induced transactivator activity in PAI-2-expressing U937 cells. This site specifically binds FosB in PAI-2-expressing U937 cells but not in HeLa cells that do not express PAI-2, and overexpression of FosB, c-Fos, or c-Jun in HeLa cells is sufficient to cause derepression of transcription from the SERPINB2 promoter. Although FosB is likely to be involved in transactivator-mediated derepression of PAI-2 transcription in macrophage-like cells, as exemplified by the U937 cell line, c-Jun may be functional in other cell types. These data suggest a model for the transcriptional control of the human PAI-2 gene and further our understanding of the molecular basis for its tissue-specific expression.
Assuntos
Modelos Biológicos , Inibidor 2 de Ativador de Plasminogênio/biossíntese , Elementos de Resposta/fisiologia , Elementos Silenciadores Transcricionais/fisiologia , Transcrição Gênica/fisiologia , Ativação Transcricional/fisiologia , Carcinógenos/farmacologia , Ativadores de Enzimas/farmacologia , Células HeLa , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Células U937RESUMO
The BZLF1 gene controls the switch between latent and lytic infection by Epstein-Barr virus (EBV). We previously reported that both the ZV and ZIIR elements within the BZLF1 promoter, Zp, are potent transcription silencers within the context of an intact EBV genome. We report here identification of another sequence element, ZV', which synergized with ZV in repressing Zp via binding ZEB1 or ZEB2. We then determined the phenotype of a variant of EBV strain B95.8 in which the ZV, ZV', and ZIIR elements were concurrently mutated. HEK293 cell lines infected with this triple mutant (tmt) virus spontaneously synthesized 6- to 10-fold more viral BZLF1, BRLF1, BMRF1, and BLLF1 RNAs, 3- to 6-fold more viral Zta, Rta, and EAD proteins, 3- to 5-fold more viral DNA, and 7- to 9-fold more infectious virus than did 293 cell lines latently infected with either the ZV ZV' double mutant (dmt) or ZIIR mutant (mt) virus. While ZV ZV' ZIIR tmt EBV efficiently infected human primary blood B cells in vitro, it was highly defective in immortalizing them. Instead of the nearly complete silencing of BZLF1 gene expression that occurs within 4 days after primary infection with wild-type EBV, the ZV ZV' ZIIR tmt-infected cells continued to synthesize BZLF1 RNA, with 90% of them dying within 9 days postinfection. BL41 cells infected with this "superlytic" virus also exhibited increased synthesis of BZLF1 and BMRF1 RNAs. Thus, we conclude that the ZV, ZV', and ZIIR silencing elements act synergistically to repress transcription from Zp, thereby tightly controlling BZLF1 gene expression, which is crucial for establishing and maintaining EBV latency.
Assuntos
Linfócitos B/metabolismo , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/fisiologia , Transativadores/biossíntese , Antígenos Virais/biossíntese , Antígenos Virais/genética , Linfócitos B/patologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Genoma Viral/fisiologia , Células HeLa , Humanos , Mutação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Viral/biossíntese , RNA Viral/genética , Elementos Silenciadores Transcricionais/fisiologia , Fatores de Tempo , Transativadores/genética , Transcrição Gênica/fisiologia , Latência Viral/fisiologiaRESUMO
Pectin is a main component of the plant cell wall and is the most complex family of polysaccharides in nature. Its composition is essential for the normal growth and morphology pattern, as demonstrated by pectin-defective mutant phenotypes. Besides this basic role in plant physiology, in tomato, pectin structure contributes to very important quality traits such as fruit firmness. Sixty-seven different enzymatic activities have been suggested to be required for pectin biosynthesis, but only a few genes have been identified and studied so far. This study characterized the tomato galacturonosyltransferase (GAUT) family and performed a detailed functional study of the GAUT4 gene. The tomato genome harbours all genes orthologous to those described previously in Arabidopsis thaliana, and a transcriptional profile revealed that the GAUT4 gene was expressed at higher levels in developing organs. GAUT4-silenced tomato plants exhibited an increment in vegetative biomass associated with palisade parenchyma enlargement. Silenced fruits showed an altered pectin composition and accumulated less starch along with a reduced amount of pectin, which coincided with an increase in firmness. Moreover, the harvest index was dramatically reduced as a consequence of the reduction in the fruit weight and number. Altogether, these results suggest that, beyond its role in pectin biosynthesis, GAUT4 interferes with carbon metabolism, partitioning, and allocation. Hence, this cell-wall-related gene seems to be key in determining plant growth and fruit production in tomato.
Assuntos
Pectinas/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Ácido Ascórbico/metabolismo , Parede Celular/química , Clonagem Molecular , Frutas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Genes de Plantas/genética , Genes de Plantas/fisiologia , Solanum lycopersicum/química , Solanum lycopersicum/genética , Solanum lycopersicum/fisiologia , Microscopia Confocal , Pectinas/análise , Fotossíntese/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Elementos Silenciadores Transcricionais/genética , Elementos Silenciadores Transcricionais/fisiologia , Ácidos Urônicos/metabolismoRESUMO
The spatially restricted expression of cadherin-7 to the intermediate domain of the neural epithelium and in migrating neural crest cells during early neural development is potentially regulated by multiple signaling inputs. To identify the regulatory modules involved in regulation of cadherin-7, evolutionary conserved non-coding sequences in the cadherin-7 locus were analyzed. This led to the identification of an evolutionary conserved region of 606 bp (ECR1) that together with the cadherin-7 promoter recapitulates endogenous cadherin-7 expression in intermediate neural tube, spinal motor neurons, interneurons, and dorsal root ganglia. Deletion analysis of ECR1 revealed a 19-bp block that is essential for ECR1 enhancer activity, while two separate blocks of 10 and 12 bp were found to be essential for ECR1 silencer activity in the dorsal and ventral neural epithelium, respectively. Together, these data provide an insight into tissue-specific regulatory regions that might be involved in regulation of cadherin-7 gene expression.
Assuntos
Caderinas/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Elementos Silenciadores Transcricionais/fisiologia , Animais , Caderinas/genética , Embrião de Galinha , Galinhas , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Elementos Silenciadores Transcricionais/genéticaRESUMO
Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular co-localization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding to nuclear DNA. In this format we can test a number of PTGS agents for cellular toxicity relative to control elements. We also developed an HTS 96-well plate assay that allows us to assess the impact of any given PTGS agent on stimulating a variety of common cellular stress signaling pathways (e.g. CRE, SRE, AP-1, NFκB, Myc, and NFAT) that could indicate possible deleterious effects of PTGS agents either dependent or independent of base pairing complementarity with target mRNAs. To this end we exploited the secreted alkaline phosphatase (SEAP) Pathway Profiling System where the expression of the secreted reporter protein is coupled to transcriptional activation of a variety of promoter elements involved in common cell signaling pathways. We found that a variety of lead hammerhead ribozyme (hhRz) and short hairpin (shRNA) expression constructs did not exert cytotoxicity in human cells when driven by highly active RNA Pol-III promoters. We also found that most of the cell signaling pathways tested (CRE, SRE, Myc, and NFAT) did not significantly couple through upregulation to expression of the set of PTGS agents tested. AP-1 and NFκB upregulation both appear to couple to the expression of some PTGS agents which likely reflect the known properties of these pathways to be stimulated by abundant small structured RNAs.
Assuntos
Ensaios de Triagem em Larga Escala , Interferência de RNA/fisiologia , Vetores Genéticos , Células HEK293/efeitos dos fármacos , Humanos , Plasmídeos , RNA/genética , RNA Catalítico/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores Depuradores Classe A/genética , Elementos Silenciadores Transcricionais/fisiologia , Ativação Transcricional , TransfecçãoRESUMO
BACKGROUND: High-throughput gene-based platform studies in human postmortem substantia nigra (SN) from sporadic Parkinson's disease (PD) cases have revealed significant dysregulation of genes involved in biological processes linked to previously established neurodegenerative mechanisms in both sporadic and hereditary PD. OBJECTIVE: Our study aimed to develop a new genetic model of PD by modulating the expression of single genes that were found to be most significantly affected in SN of sporadic PD. METHODS: SN-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses carrying different gene-specific sequences. RESULTS: Silencing of the E3 ligase ubiquitin SKP1A resulted in a decline in the expression of dopaminergic phenotypic markers together with progression into an aberrant cell cycle and death. Furthermore, added knockout of the dopamine-metabolizing enzyme aldehyde dehydrogenase, found almost absent in sporadic PD SN pars compacta, exacerbated the vulnerability of SKP1A-silenced neurons to MPP(+) and neurotrophin deprivation. CONCLUSION: Future studies should focus on a careful consideration of crucial dopaminergic gene network interactions as emerged from human sporadic PD, which will serve as a basis for the development of a slowly progressive genetic animal model of sporadic PD, with the potential of evaluating drugs with 'disease-modifying activity'.
Assuntos
Modelos Animais de Doenças , Predisposição Genética para Doença , Doença de Parkinson/genética , Interferência de RNA/fisiologia , Animais , Humanos , Modelos Biológicos , Doença de Parkinson/etiologia , Doença de Parkinson/patologia , Elementos Silenciadores Transcricionais/genética , Elementos Silenciadores Transcricionais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Insulators are cis-regulatory elements that prevent improper gene activation and heterochromatin spreading. An insulator is contained in the Mcp boundary from the regulatory region of the Drosophila melanogaster Abd-B gene. The barrier function of the Mcp insulator was studied and proved to be due to two modules. One is responsible for long-distance interactions and the capability of blocking enhancers. The other is essential for blocking Pc-dependent repression. It was observed for the first time that an insulator increased the repressor activity of a neighbor silencer.
Assuntos
Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Elementos Isolantes/fisiologia , Elementos Silenciadores Transcricionais/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Heterocromatina/genética , Proteínas de Homeodomínio/genéticaRESUMO
Inducible defenses against oxidative stress are coordinated by redox-sensitive transcription factors that transduce oxidative damage into differential gene expression. The opportunistic human pathogen Pseudomonas aeruginosa has evolved under physiological and host-derived sources of oxidative stress. Previous work showed that the pqrABC and pqrR genes of P. aeruginosa, all lacking known functions, were induced by treatment of three different isolates of P. aeruginosa with paraquat (PQ), a superoxide-producing agent. Insertional mutation of the pqrABCR genes resulted in hypersensitive phenotypes to a variety of oxidants, although the hypersensitivity to PQ was marginal. Mutation of pqrR and complementation assays showed that PqrR regulated the pqrABC genes in response to PQ. PqrR, a member of the MarR family of transcriptional regulators, contains a C-terminal region with four conserved cysteines, which suggested redox-regulated transcriptional activity. Purified PqrR bound to two discrete sites at the pqrA and pqrR regulatory regions. The in vitro DNA binding activity of PqrR was decreased by exposure to air and reconstituted by treatment with dl-dithiothreitol. Elemental analysis and preliminary electron paramagnetic resonance experiments showed that PqrR contains iron. Interestingly, site-directed mutagenesis of C-terminal cysteines demonstrated that the four conserved cysteine residues are essential for in vivo redox sensing by PqrR.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/química , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais/fisiologia , Elementos Silenciadores Transcricionais/fisiologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Diamida/farmacologia , Dados de Sequência Molecular , Mutação , Naftoquinonas/farmacologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Paraquat/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Transcrição GênicaRESUMO
DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate the cis-acting responses to DSBs in G1 phase cells. We found that DSBs within a gene body silence its expression, as well as the transcription of local undamaged genes at a distance defined by the spread of γ-H2AX from the DSB. Importantly, DSBs not only repress ongoing transcription but also block the inducible expression of regional genes. DSB-mediated transcriptional repression depends on DDR signaling but does not require the generation of inaccessible chromatin. Our findings demonstrate that in G1 phase cells, DDR signaling establishes a robust and extensive region of transcriptional repression spreading from DSB sites and introduce an approach to study the mechanistic impact of targeted DNA breaks in nearly any chromatin environment.
Assuntos
Reparo do DNA/genética , Fase G1/genética , Elementos Silenciadores Transcricionais/genética , Animais , Ciclo Celular/genética , Linhagem Celular , DNA/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/fisiologia , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Humanos , Camundongos , Elementos Reguladores de Transcrição/genética , Elementos Reguladores de Transcrição/fisiologia , Elementos Silenciadores Transcricionais/fisiologiaRESUMO
An intronic silencer, S4, in the Cd4 gene has been shown to be responsible for the helper-lineage-specific expression of CD4; S4 requires Runx complex binding to exert its silencer function against the enhancer-mediated Cd4 activation by modulating the epigenetic state of the Cd4 gene. Here we identify a late-acting maturation enhancer. Bcl11b plays essential roles for activation of both the early-acting proximal enhancer and maturation enhancer of Cd4. Notably, Runx complexes suppress these enhancers by distinct mechanisms. Whereas repression of the proximal enhancer depends on the S4 silencer, the maturation enhancer is repressed by Runx in the absence of S4. Moreover, ThPOK, known to antagonize S4-mediated Cd4 repression, assists Runx complexes to restrain maturation enhancer activation. Distinct modes of S4 silencer action upon distinct enhancers thus unravel a pathway that restricts CD4 expression to helper-lineage cells by silencer-independent and Runx-dependent repression of maturation enhancer activity in cytotoxic-lineage cells.
Assuntos
Antígenos CD4/genética , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Repressoras/metabolismo , Linfócitos T Auxiliares-Indutores/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Antígenos CD4/metabolismo , Células Cultivadas , Quimera , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Íntrons/genética , Camundongos , Camundongos Transgênicos , Proteínas Repressoras/genética , Elementos Silenciadores Transcricionais/fisiologia , Linfócitos T Citotóxicos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
In Saccharomyces cerevisiae, silencers flanking the HML and HMR loci initiate the establishment of transcriptional silencing. We demonstrate that the activity of a silencer pertaining to its potency and directionality is dependent on its genomic position. The context of the HML-E silencer is more permissive to silencer function than that of HML-I or HMR-E, despite that HML-E and HML-I are only 3.3 kb apart. The apparent strength and directionality of a silencer in a particular location is affected by other silencing elements (silencers and protosilencers) present in its context. We show that at the HML locus, at least four silencing elements engage in multiple functional interactions that contribute to the activities of the silencers. Notably, these dispersed silencing elements can synergize to silence genes located not only inside, but also outside the HML sequence that harbors them. Moreover, the relative positions and orientations of these elements are important for silencing, indicating that they belong to an intricate silencing network.
Assuntos
Ordem dos Genes/fisiologia , Inativação Gênica/fisiologia , Saccharomyces cerevisiae/genética , Elementos Silenciadores Transcricionais/fisiologia , Técnicas Genéticas , Genoma Fúngico , Modelos Biológicos , Origem de Replicação/fisiologia , Telômero/fisiologiaRESUMO
The increasing data show that a parallel G-quadruplex structure formed by the element of nuclease-hypersensitivity element III(1) (NHE III(1)) in the P1 promoter of c-myc functions as a transcriptional repressor. Triethylene tetraamine has exhibited interesting properties of stabilizing both inter- and intra-molecular G-quadruplex structures and telomerase-inhibitory potency. Here, we present evidences showing that triethylene tetraamine facilitates the formation of G-quadruplex structure by the NHE III(1) element, and inhibits the expression of c-myc in HeLa cells.
Assuntos
DNA/efeitos dos fármacos , Genes myc , Regiões Promotoras Genéticas/efeitos dos fármacos , Elementos Silenciadores Transcricionais/fisiologia , Trientina/farmacologia , Regulação para Baixo , Quadruplex G , Guanina/química , Células HeLa , Humanos , Proteínas Proto-Oncogênicas c-myc/biossínteseRESUMO
Runx family proteins have the potential for either activating or suppressing gene expression in a context-dependent manner. There are several mechanisms by which transcriptional repression can occur. A wide range of locus inactivation, that is often called gene silencing, is thought to be achieved by chromatin modifications. Recently, Runx family proteins were found to have an essential role in either temporal transcriptional repression or irreversible epigenetic silencing at the CD4 locus through binding to a CD4 silencer at different stages of development. These findings link Runx function to epigenetic gene regulation, and shed new light on the mechanisms by which Runx represses target gene expression.
Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética , Inativação Gênica , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Animais , Antígenos CD4/genética , Subunidade alfa 2 de Fator de Ligação ao Core , Subunidade alfa 3 de Fator de Ligação ao Core , Subunidades alfa de Fatores de Ligação ao Core , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/etiologia , Neoplasias/genética , Proteínas Proto-Oncogênicas/metabolismo , Elementos Silenciadores Transcricionais/genética , Elementos Silenciadores Transcricionais/fisiologia , Fatores de Transcrição/metabolismoAssuntos
Córtex Cerebral/fisiopatologia , Sistema Límbico/fisiopatologia , Estado Epiléptico/fisiopatologia , Animais , Estimulação Elétrica , Eletroencefalografia , Humanos , Modelos Neurológicos , Elementos Silenciadores Transcricionais/fisiologia , Substância P/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Chromatin insulators, which define domains of transcriptional autonomy, are cis-acting elements of eukaryotes. They are located at the boundaries of differentially regulated genes and delimit their interactions by establishing independent chromatin structures. They can block chromosomal enhancers or silencers if they are located between promoters and enhancers or silencers, and protect against position effect. Recent studies focus on elucidating the molecular mechanism of insulator functions. Loop Model is the most predominant hypothesis while others are assumed. Study progress in this field sheds new light on the regulation of gene expression.
Assuntos
Cromatina/fisiologia , Regulação da Expressão Gênica , Elementos Isolantes/fisiologia , Animais , Fator de Ligação a CCCTC , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Humanos , Elementos Isolantes/genética , Proteínas Repressoras/metabolismo , Elementos Silenciadores Transcricionais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3' splice sites by binding 5'-UAG-3' elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A(+)C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5'-UAGU-3' element and a proximal 5'-GAU-3' motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5'-GAU-3' and 5'-UAGU-3' motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K(d)=37.8±1.1 nM) complex with ESS3 via site-specific interactions with the loop.
Assuntos
Processamento Alternativo , HIV-1/genética , Ribonucleoproteínas Nucleares Heterogêneas/química , RNA Viral/química , Elementos Silenciadores Transcricionais/genética , Processamento Alternativo/genética , Éxons/genética , HIV-1/química , HIV-1/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Sítios de Splice de RNA/genética , RNA Viral/metabolismo , Elementos Silenciadores Transcricionais/fisiologia , Soluções/química , TermodinâmicaRESUMO
BACKGROUND: The human cervical cancer oncogene HCCR-2 is overexpressed in various malignant tumors and cell lines, and might function as a negative regulator of the p53 tumor suppressor. Here, we used RNA interference strategies to evaluate the role of HCCR-2 in liver cancer, and to explore its potential therapeutic effect. METHODS: Changes of HepG2 cells stably transfected by an HCCR-2 RNA interference vector were detected by real-time PCR, MTT staining, plate colony formation, flow cytometry, and cell migration experiments. Apoptosis-related protein Bcl-2 and Bax levels were measured by Western blot. RESULTS: Our results showed that of the three siRNA-expressing vectors, siRNA-H3 had a suppressive effect on the expression of HCCR-2 mRNA, interfering with proliferation and migration of HCCR-2. Moreover, the apoptotic rate also increased, and cells transfected by siRNA-H3 were blocked in the G0/G1 stage. Plate colony formation experiments demonstrated that the single cell clone formation capacity of HepG2-H3 cells was clearly lower than that of HepG2 and HepG2-N cells. Western blot results indicated that the expression of Bcl-2 was inhibited, and the expression of Bax was increased. CONCLUSIONS: In summary, RNAi targeting HCCR-2 could be an effective means for suppressing malignant features of hepatocellular carcinoma cells.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas/fisiologia , Elementos Silenciadores Transcricionais/fisiologia , Apoptose , Técnicas de Cultura de Células , Proliferação de Células , Células Hep G2 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/metabolismoRESUMO
Transcriptional repression is essential for establishing precise patterns of gene expression during development. Repressors governing early Drosophila segmentation can be classified as short- or long-range factors based on their ranges of action, acting either locally to quench adjacent activators or broadly to silence an entire locus. Paradoxically, these repressors recruit common corepressors, Groucho and CtBP, despite their different ranges of repression. To reveal the mechanisms underlying these two distinct modes of repression, we performed chromatin analysis using the prototypical long-range repressor Hairy and the short-range repressor Knirps. Chromatin immunoprecipitation and micrococcal nuclease mapping studies reveal that Knirps causes local changes of histone density and acetylation, and the inhibition of activator recruitment, without affecting the recruitment of basal transcriptional machinery. In contrast, Hairy induces widespread histone deacetylation and inhibits the recruitment of basal machinery without inducing chromatin compaction. Our study provides detailed mechanistic insight into short- and long-range repression on selected endogenous target genes and suggests that the transcriptional corepressors can be differentially deployed to mediate chromatin changes in a context-dependent manner.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Elementos Silenciadores Transcricionais/genética , Acetilação , Oxirredutases do Álcool/metabolismo , Animais , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Nuclease do Micrococo/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Elementos Silenciadores Transcricionais/fisiologiaRESUMO
The transforming growth factor beta (TGF-beta) family member Decapentaplegic (Dpp) is a key regulator of patterning and growth in Drosophila development. Previous studies have identified a short DNA motif called the silencer element (SE), which recruits a trimeric Smad complex and the repressor Schnurri to downregulate target enhancers upon Dpp signaling. We have now isolated the minimal enhancer of the dad gene and discovered a short motif we termed the activating element (AE). The AE is similar to the SE and recruits the Smad proteins via a conserved mechanism. However, the AE and SE differ at important nucleotide positions. As a consequence, the AE does not recruit Schnurri but rather integrates repressive input by the default repressor Brinker and activating input by the Smad signal transducers Mothers against Dpp (Mad) and Medea via competitive DNA binding. The AE allows the identification of hitherto unknown direct Dpp targets and is functionally conserved in vertebrates.