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Pichia pastoris (Komagataella phaffii) is a fast-growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co-express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed-batch modes. First, the production of phytase was investigated under glucose de-repression in continuous culture at three dilution factors, 0.5 d-1 , 1 d-1 , and 1.5 d-1 . The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed-batch modes for growth and phytase production. The kinetic model developed aided to design the glucose-feeding profile of a fed-batch culture. Kinetic model simulations under glucose de-repression and fed-batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h-1 . Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de-repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5-fold by methanol induction compared to the expression with glucose alone under de-repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36-fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.
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BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica. RESULTS: This study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter's strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use. CONCLUSIONS: In this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.
Assuntos
Yarrowia , Yarrowia/metabolismo , Eritritol/metabolismo , Biologia Sintética , Regiões Promotoras Genéticas , Engenharia MetabólicaRESUMO
Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1ß, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway.
Assuntos
Regulação da Expressão Gênica , MAP Quinase Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , RNA Longo não Codificante/metabolismo , Vasculite/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , MAP Quinase Quinase Quinase 4/genética , Camundongos , RNA Longo não Codificante/genética , Vasculite/genética , Vasculite/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing ß-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.
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Engenharia Metabólica , Zea mays , Animais , Galinhas , Plantas Geneticamente Modificadas/genética , Xantofilas , Zea mays/genéticaRESUMO
Bidirectional promoters are the transcription regulatory regions of genes positioned head-to-head on opposite strands. Specific sequence signals, chromatin modifications and three-dimensional structures of the transcription site facilitate the unconventional yet tightly regulated transcription proceeding in both directions from these promoters. Mutations or aberrant epigenetic changes can lead to abnormal enhanced or reduced expression from either of the bidirectionally transcribed genes resulting in tumorigenesis. Moreover, bidirectionally transcribed genes might also contribute towards the immune regulation in tumor microenvironment. In this review, we aimed to expound the characteristic features of bidirectional promoters alongside their transcriptional regulations, and ultimately, the association of these enigmatic genomic elements in different cancers.
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Regulação Neoplásica da Expressão Gênica , Genoma Humano , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Transformação Celular Neoplásica/genética , Ilhas de CpG/genética , Epigênese Genética , Humanos , Transcrição Gênica , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
We conducted an integrated analysis of gene expression and chromatin structure of mouse uterus to understand the regulation of uterine-expressed genes on gestation day 4 (GD4) during the peri-implantation period. The genes expressed in the uterus showed a significant association (p < .0001) with the presence of the nucleosome-free region (open chromatin) in the 5'-untranslated region of the genes. The majority of these upstream open chromatins harbored a common class of regulatory elements known as upstream open reading frames. We also compared the gene expression profiles between the uterus and brain which showed that specific gene pairs were expressed in a correlated manner, either positively or negatively. In addition, specific ligand/receptor genes showed coordinated patterns of expression between the uterus and brain on GD4, and the level of expression of these ligand/receptors altered significantly in the brain during late pregnancy (GD15) compared with the peri-implantation period (GD4). Collectively, these results suggest that regulation of the uterine genes during the peri-implantation period is likely to have a functional link with the maternal brain in pregnant mice.
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Encéfalo/metabolismo , Implantação do Embrião/genética , Regulação da Expressão Gênica , Idade Gestacional , Útero/metabolismo , Regiões 5' não Traduzidas , Animais , Cromatina/genética , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Mensageiro/genética , TranscriptomaRESUMO
A locus on chromosome 9q22 harbors a SNP (rs965513) firmly associated with risk of papillary thyroid carcinoma (PTC). The locus also comprises the forkhead box E1 (FOXE1) gene, which is implicated in thyroid development, and a long noncoding RNA (lncRNA) gene, papillary thyroid cancer susceptibility candidate 2 (PTCSC2). How these might interact is not known. Here we report that PTCSC2 binds myosin-9 (MYH9). In a bidirectional promoter shared by FOXE1 and PTCSC2, MYH9 inhibits the promoter activity in both directions. This inhibition can be reversed by PTCSC2, which acts as a suppressor. RNA knockdown of FOXE1 in primary thyroid cells profoundly interferes with the p53 pathway. We propose that the interaction between the lncRNA, its binding protein MYH9, and the coding gene FOXE1 underlies the predisposition to PTC triggered by rs965513.
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Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Fatores de Transcrição Forkhead/genética , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 9/genética , Fatores de Transcrição Forkhead/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Câncer Papilífero da TireoideRESUMO
The human genome harbors many duplicated segments, which sometimes show very high sequence identity. This may complicate assignment during genome assembly. One such example is in Xq28, where the arrangement of 2 recently duplicated segments varies between genome assembly versions. The duplicated segments comprise highly similar genes, including MAGEA3 and MAGEA6, which display specific expression in testicular germline cells, and also become aberrantly activated in a variety of tumors. Recently, a new gene was identified, CT-GABRA3, the transcription of which initiates inside the segmental duplication but extends far outside. According to the latest genome annotation, CT- GABRA3 starts near MAGEA3, with which it shares a bidirectional promoter. In an earlier annotation, however, the duplicated segment was positioned in the opposite orientation, and CT-GABRA3 was instead coupled with MAGEA6. To resolve this discrepancy, and based on the contention that genes connected by a bidirectional promoter are almost always co-expressed, we decided to compare the expression profiles of CT-GABRA3, MAGEA3, and MAGEA6. We found that in tumor tissues and cell lines of different origins, the expression of CT-GABRA3 was better correlated with that of MAGEA6. Moreover, in a cellular model of experimental induction with a DNA demethylation agent, activation CT-GABRA3 was associated with that of MAGEA6, but not with that of MAGEA3. Together these results support a connection between CT-GABRA3 and MAGEA6 and illustrate how promoter-sharing genes can be exploited to resolve genome assembly uncertainties.
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Antígenos de Neoplasias/genética , Cromossomos Humanos X/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Receptores de GABA-A/genética , Duplicações Segmentares Genômicas/genética , Antígenos de Neoplasias/metabolismo , Epigênese Genética/genética , Duplicação Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Heart development is a relatively fragile process in which many transcription factor genes show dose-sensitive characteristics such as haploinsufficiency and lower penetrance. Despite efforts to unravel the genetic mechanism for overcoming the fragility under normal conditions, our understanding still remains in its infancy. Recent studies on the regulatory mechanisms governing gene expression in mammals have revealed that long non-coding RNAs (lncRNAs) are important modulators at the transcriptional and translational levels. Based on the hypothesis that lncRNAs also play important roles in mouse heart development, we attempted to comprehensively identify lncRNAs by comparing the embryonic and adult mouse heart and brain. RESULTS: We have identified spliced lncRNAs that are expressed during development and found that lncRNAs that are expressed in the heart but not in the brain are located close to genes that are important for heart development. Furthermore, we found that many important cardiac transcription factor genes are located in close proximity to lncRNAs. Importantly, many of the lncRNAs are divergently transcribed from the promoter of these genes. Since the lncRNA divergently transcribed from Tbx5 is highly evolutionarily conserved, we focused on and analyzed the transcript. We found that this lncRNA exhibits a different expression pattern than that of Tbx5, and knockdown of this lncRNA leads to embryonic lethality. CONCLUSION: These results suggest that spliced lncRNAs, particularly bidirectional lncRNAs, are essential regulators of mouse heart development, potentially through the regulation of neighboring transcription factor genes.
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Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.
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Arabidopsis/metabolismo , Gossypium/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas/genética , Arabidopsis/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gossypium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genéticaRESUMO
The goat pox chick embryo-attenuated virus (GTPV) has been developed as an effective vaccine that can elicit protective immune responses. It possesses a large genome and a robust ability to express exogenous genes. Thus, this virus is an ideal vector for recombinant live vaccines for infectious diseases in ruminant animals. In this study, we identified a novel bidirectional promoter region of GTPV through screening named PbVV(±). PbVV(±) is located between ETF-l and VITF-3, which are transcribed in opposite directions. A new recombinant goat pox virus (rGTPV) was constructed, in which duplicate PbVV(+) was used as a promoter element to enhance Brucella OMP31 expression, and duplicate PbVV(-) was used as a promoter element to regulate enhanced green fluorescent protein (EGFP) at the same time as the selection marker. PbVV(-) promoter activity was compared to that of the P7.5 promoter of vaccinia virus, as measured by EGFP expression; the fluorescence intensity of EGFP expressed in cells was confirmed by fluorescence microscopy and flow cytometry. PbVV(+) promoter activity was measured by Brucella OMP31 expression. Interaction with the anti-Brucella-OMP31 monoclonal antibody was confirmed by western blotting, and OMP31 mRNA expression was assessed by qRT-PCR. The results of this study will be useful for the further study of effective multivalent vaccines based on rGTPV. This study also provides a theoretical basis for overcoming the problem of low expression of exogenous genes.
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Capripoxvirus/genética , Regiões Promotoras Genéticas , Animais , Proteínas da Membrana Bacteriana Externa/genética , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genéticaRESUMO
We previously identified proline-rich protein 11 (PRR11) as a novel cancer-related gene that is implicated in the regulation of cell cycle and tumorigenesis. Our recent study demonstrated that PRR11 and its adjacent gene, kinetochore associated 2 (SKA2), constitute a classic head-to-head gene pair that is coordinately regulated by nuclear factor Y (NF-Y). In the present study, we further show that the PRR11-SKA2 bidirectional transcription unit is an indirect target of the tumor suppressor p53. A luciferase reporter assay revealed that overexpression of wild type p53, but not mutant p53, significantly represses the basal activity and NF-Y mediated transactivation of the PRR11-SKA2 bidirectional promoter. Deletion and mutation analysis of the PRR11-SKA2 promoter revealed that p53-mediated PRR11-SKA2 repression is dependent on the presence of functional NF-Y binding sites. Furthermore, a co-immunoprecipitation assay revealed that p53 associates with NF-Y in lung cancer cells, and a chromatin immunoprecipitation assay showed that p53 represses PRR11-SKA2 transcription by reducing the binding amount of NF-Y in the PRR11-SKA2 promoter region. Consistently, the ability of p53 to downregulate PRR11-SKA2 transcription was significantly attenuated upon siRNA-mediated depletion of nuclear factor Y subunit beta (NF-YB). Notably, lung cancer patients with lower expression of either PRR11 or SKA2 along with wild type p53 exhibited the best overall survival compared with others with p53 mutation and/or higher expression of either PRR11 or SKA2. Taken together, our results demonstrate that p53 negatively regulates the expression of the PRR11-SKA2 bidirectional transcription unit through NF-Y, suggesting that the inability to repress the PRR11-SKA2 bidirectional transcription unit after loss of p53 might contribute to tumorigenesis.
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Fator de Ligação a CCAAT/metabolismo , Proteínas Cromossômicas não Histona/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/mortalidade , Prognóstico , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Head-to-head gene pairs represent a unique feature of gene organization in eukaryotes, accounting for >10% of genes in the human genome. Identification and functional analysis of such gene pairs is only in its infancy. Recently, we identified PRR11 as a novel cancer-related gene that is implicated in cell cycle and lung cancer. Here we demonstrate that PRR11 is oriented in a head-to-head configuration with its neighboring gene, SKA2. 5'-RACE assay revealed that the intergenic spacer region between the two genes is <500 bp. Serial luciferase reporter assays demonstrated that a minimal 80-bp intergenic region functions as a core bidirectional promoter to drive basal transcription in both the PRR11 and SKA2 orientations. EMSA and ChIP assays demonstrated that NF-Y binds to and directly transactivates the PRR11-SKA2 bidirectional promoter. SiRNA-mediated NF-Y depletion significantly downregulated PRR11 and SKA2 expression. Expression of both PRR11 and SKA2 was significantly upregulated in lung cancer. Expression of the two genes was highly correlated with each other and with NF-Y expression. Remarkably, high expression of both PRR11 and SKA2 was associated with poorer prognosis in lung cancer patients compared with high expression of one gene or low expression of both genes. Knockdown of PRR11 and/or SKA2 remarkably reduced cell proliferation, migration, and invasion in lung cancer cells. Thus, the PRR11-SKA2 bidirectional transcription unit, which is a novel direct target of NF-Y, is essential for the accelerated proliferation and motility of lung cancer cells and may represent a potential target in the diagnosis and/or treatment of human lung cancer.
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Fator de Ligação a CCAAT/fisiologia , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas , Proteínas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cromatina/genética , DNA/genética , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
BACKGROUND: In the presence of sufficient iron, the Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction. RESULTS: We cloned the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies. CONCLUSIONS: The vectors pFCF1, pFCF2, and pFBH1 have been shown to use the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Vetores Genéticos/genética , Ferro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular anomalies of the nervous system mostly located in the brain presenting sporadically or familial. Causes of familial forms are mutations in CCM1 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10) genes. Sporadic forms with no affected relative most often have only one lesion and no germ line mutations. However, a number of sporadic cases with multiple lesions have been reported and are indeed genetic cases with a de novo mutation or a mutation inherited from an asymptomatic parent. METHODS: Here, we performed an analysis of regulatory region of CCM genes in 60 sporadic patients, negative for mutations in coding region and intron-exon boundaries and large deletion/duplications in CCM genes by direct sequencing and MLPA. Among 5 variants identified in 851-bp region shared by CCM3 and SERPINI1 genes and acting as asymmetric bidirectional promoter, two polymorphisms c.-639 T > C/rs9853967 and c.-591 T > C/rs11714980 were selected. A case-control study was performed to analyze their possible relationships with sporadic CCMs. Promoter haplotypes activities on CCM3/SERPINI1 genes expression were tested by dual-luciferase assay. RESULTS: No variants were identified in CCM1 and CCM2 regulatory regions. In CCM3/SERPINI1 asymmetric bidirectional promoter 5 variants, 2 of them unknown and 3 corresponding to polymorphisms c.-639 T > C/rs9853967, c.-591 T > C/rs11714980 and c.-359G > A/rs9834676 were detected. While rs9853967 and rs11714980 polymorphisms fall in a critical regulatory fragment outside the minimal promoter in intergenic region, other variants had no effects on transcription factor binding according to RegRNA tool. Case-control study performed on 60 patients and 350 healthy controls showed frequencies of the mutated alleles significantly higher in the control group than in patients. Furthermore, the functional assay showed a significant reduction of CCM3 expression for C-C haplotype even more than for T-C and C-T haplotypes. In SERPINI1 direction, the reduction was not statistically significant. CONCLUSIONS: Our data indicated that rs9853967 and rs11714980 polymorphisms could be associated with a protective role in CCM disease.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas de Membrana/genética , Neuropeptídeos/genética , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Serpinas/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Adulto Jovem , NeuroserpinaRESUMO
Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or ß-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters.
Assuntos
DNA Intergênico/fisiologia , Genes de Plantas/genética , Proteínas de Plantas/fisiologia , Regiões Promotoras Genéticas/fisiologia , Zea mays/genética , DNA Intergênico/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Sementes/metabolismo , Sementes/fisiologia , Transcriptoma , Zea mays/fisiologiaRESUMO
KEY MESSAGE: The bidirectional promoter of the Arabidopsis thaliana gene pair At1g71850/At1g71860 harbors low-temperature-responsive elements, which participate in anti-correlated transcription regulation of the driving genes in response to environmental low temperature. A divergent gene pair is defined as two adjacent genes organized head to head in opposite orientation, sharing a common promoter region. Divergent gene pairs are mainly coexpressed, but some display opposite regulation. The mechanistic basis of such anti-correlated regulation is not well understood. Here, the regulation of the Arabidopsis thaliana gene pair At1g71850/At1g71860 was investigated. Semi-quantitative RT-PCR and Genevestigator analyses showed that while one of the pair was upregulated by exposure to low temperature, the same treatment downregulated the other. Promoter::GUS fusion transgenes were used to show that this behavior was driven by a bidirectional promoter, which harbored an as-1 motif, associated with the low-temperature response; mutation of this sequence produced a significant decrease in cold-responsive expression. With regard to the as-1 motif in the native orientation repressing the promoter's low-temperature responsiveness, the same as-1 motif introduced in the reverse direction showed a slight enhancement in the promoter's responsiveness to low-temperature exposure, indicating that the orientation of the motif was important for the promoter's activity. These findings provide new insights into the complex transcriptional regulation of bidirectional gene pairs as well as plant stress response.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Regiões Promotoras Genéticas , Proteínas Tirosina Fosfatases/genética , Ubiquitina Tiolesterase/genética , Proteínas de Arabidopsis/metabolismo , Fluorometria , Glucuronidase/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Estresse Fisiológico/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Many mammalian genes are composed of bidirectional gene pairs with the two genes separated by less than 1.0kb. The transcriptional regulation and function of these bidirectional genes remain largely unclear. Here, we report that bidirectional gene pair HspB2/αB-crystallin, both of which are members of the small heat shock protein gene family, is a novel direct target gene of p53. Two potential binding sites of p53 are present in the intergenic region of HspB2/αB-crystallin. p53 up-regulated the bidirectional promoter activities of HspB2/αB-crystallin. Actinomycin D (ActD), an activator of p53, induces the promoter and protein activities of HspB2/αB-crystallin. p53 binds to two p53 binding sites in the intergenic region of HspB2/αB-crystallin in vitro and in vivo. Moreover, the products of bidirectional gene pair HspB2/αB-crystallin regulate glucose metabolism, intracellular reactive oxygen species (ROS) level and the Warburg effect by affecting metabolic genes, including the synthesis of cytochrome c oxidase 2 (SCO2), hexokinase II (HK2), and TP53-induced glycolysis and apoptosis regulator (TIGAR). The ROS level and the Warburg effect are affected after the depletion of p53, HspB2 and αB-crystallin respectively. Finally, we show that both HspB2 and αB-crystallin are linked with human renal carcinogenesis. These findings provide novel insights into the role of p53 as a regulator of bidirectional gene pair HspB2/αB-crystallin-mediated ROS and the Warburg effect.
Assuntos
Carcinoma de Células Renais/genética , Proteínas de Choque Térmico HSP27/genética , Proteína Supressora de Tumor p53/genética , Cadeia B de alfa-Cristalina/genética , Sítios de Ligação/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Regiões Promotoras Genéticas , Ligação Proteica/genética , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
In the silk moth Bombyx mori, chorion genes of the same developmental specificity are organized in divergently transcribed α/ß gene pairs, sharing a common 5' flanking promoter region. This bidirectional promoter contains a complete set of cis-elements responsible for developmentally accurate gene expression. In the present paper, based on the observation that Bombyx chorion gene promoters contain cis-elements for the same transcription factors without concrete evidence on which of them are essential, we address the question as to how promoter architecture (number, orientation and position of common factor binding sites) facilitates developmentally accurate chorion gene regulation. To this end, we constructed several mutated promoter regions of an early-middle gene pair and cloned them upstream of a reporter gene to introduce these plasmid constructs into silk moth follicle epithelial cells via electroporation as an efficient and quick method for transient expression. This is the first time that an ex vivo method had been applied to test the impact of systematic cis-element mutations on a chorion gene promoter. Our results confirmed the importance of the HMGA factor and the role of the GATA factor as an early repressor, and led to a more detailed understanding of which C/EBP sites participate in the regulation of early-middle chorion gene expression.
Assuntos
Bombyx/genética , Córion/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/fisiologia , Animais , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Eletroporação , Feminino , Fatores de Transcrição GATA/metabolismo , Proteínas HMGA/metabolismo , Mutagênese Sítio-Dirigida , Fatores de TranscriçãoRESUMO
Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci.