Chimeric Promoter Research Articles

Chimeric liver specific promoters for long-term gene expression in the liver

Chimeric liver specific promoters for long-term gene expression in the liver

Developmental control of the cell cycle.

Whereas animal development has a defined and early embryonic phase during which all of the major organ systems are put in place, plant growth continues throughout the life of the organism. Moreover, most of the above ground plant growth that we see is modular and the modules, known as phytomers, are formed by the activity of shoot apical meristem. This results in at least two distinct types of growth: indeterminate growth as shown by the meristem, producing tissues of undefined maximum size; and determinate growth as shown by leaves and other lateral organs, producing organs of limited maximum size. We are interested in how plant cell proliferation is regulated within the context of development. Mutations in developmental genes can affect the pattern and the extent of cell growth or proliferation. Thus it can be argued that such genes regulate cell growth division. The molecular mechanisms by which developmental genes influence cell division and proliferation have remained largely obscure. Developmental patterning and cell cycle control appear to be co-ordinated throughout plant growth and this is reflected by temporal and spatial regulation of regulatory genes such as cyclins and cyclin dependent protein kinases. Using an approach based on RNA in situ hybridisation, we have characterised the expression patterns of a number of cell cycle genes (Fobert et al., 1994; Fobert et al., 1996; Gaudin et al., 2000). Based on this survey, many cell cycle control genes are expressed throughout the plant in a cell cycle-stage specific manner, and produce a spotty pattern (Fig. 1). Double label in situ methods (Fobert et al., 1994) and synchronised cell (Sorrell et al., 2001) studies permit the further resolution of expression periods (Fig. 1). Many of the genes, such as B-cyclins, kinesins and ICKs expressed during the G2/M phase, contain characteristic motifs, called MSA elements, within their 5# non-coding regions. Molecular analysis of these MSA elements indicates that they are necessary and sufficient to direct G2/M gene expression and that they bind to 3 repeat Myb proteins (Ito et al., 2001). The plant 3 repeat Myb proteins are similar to the mammalian cMybs that are implicated in G1/S progression. Therefore, their role has been conserved in that both animal and plant genes regulate cell cycle phase transitions, but the phase affected is different. Plant 3 repeat Myb genes form two classes, one of which appears to act positively and is up regulated during G2/M; the other acts as an inhibitor and is expressed throughout the cell cycle. The transcripts of other cell cycle related genes do not change markedly during the cell cycle. These include the PSTAIR cdk genes and cyclin A and D genes. These functions may be regulated post-transcriptionally. Gainand loss-of-function transgenic and mutant approaches are being used to dissect the function of such genes. The alcA/alcR gene switch is a two component chemically inducible system for regulating gene expression in a range of organisms. Briefly, this system involves two components, originally from the filamentous fungus, Aspergillus nidulans. The transactivator, ALCR, is activated by a treatment of the plant with a low level of ethanol and a chimeric promoter, pAlcA/35S that contains a minimal 35S promoter and the upstream regulatory sequences of the Aspergillus alcA gene containing the binding sites of ALCR. The ethanol switch is functional in tobacco and has been transferred to Arabidopsis (Roslan et al., 2001) where we are using it to up and down regulate the expression of key developmental and cell cycle regulatory genes. We have created a range of transactivator lines that express the alcR transactivator in specific domains of the meristem or leaves. These lines should be generally useful for manipulating meristem growth or development. * Corresponding author. Tel.: +44-1603-450288; fax: +44-1603-450022. E-mail address: john.doonan@bbsrc.ac.uk (J.H. Doonan). Cell Biology International 27 (2003) 283–285 Cell Biology International

Positive- and negative-acting signals combine to determine differential RNA replication from the paramyxovirus simian virus 5 genomic and antigenomic promoters

The cis-acting signals found at the 3′ ends of the genomic and antigenomic RNAs are a major factor determining the level of paramyxovirus RNA replication from each promoter. Using a minigenome system that reconstitutes SV5 RNA synthesis from cDNA-derived components, we show here that the genomic promoter (GP) for the paramyxovirus SV5 directs RNA replication ∼14-fold lower than that seen from the antigenomic promoter (AGP). The goal of this study was to identify cis-acting signals responsible for differential levels of RNA replication from the SV5 GP and AGP. We have previously shown that the SV5 AGP contains three sequence-dependent elements (CRI, CRII, and Region III) that are separated by sequence-independent spacer regions. Minigenomes containing chimeric promoters were constructed to test the hypothesis that transfer of discrete cis-acting AGP elements to the GP could confer higher replication properties to the GP. Minigenomes containing a substitution of the AGP CRI, CRII, or Region III elements alone in place of the corresponding GP sequences did not show enhanced levels of RNA replication. However, transfer of both the AGP 3′ terminal CRI and Region III elements into the corresponding sites of the GP led to a minigenome which replicated to ∼40% of the levels seen with the AGP. This enhanced RNA replication from the GP was further increased up to AGP levels by also including the intervening AGP segment (bases 20–50) located between CRI and Region III. Importantly, transfer of nonviral sequences in place of GP bases 20–50 also increased RNA replication to levels approaching that of the AGP, but only in the context of the AGP CRI and Region III substitutions. These data indicate that differential levels of RNA replication from the SV5 GP and AGP are due to a combination of positive-acting signals in the AGP (CRI and Region III) and a negative-acting signal in the GP (bases 20–50). Possible functions for the SV5 promoter elements in determining RNA replication levels are proposed.

Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system, composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.

Suppression of the ERK1/2 signaling pathway from HCV NS5A protein expressed by herpes simplex recombinant viruses.

Two herpes simplex type 1 (HSV-1) recombinant viruses carrying the hepatitis C virus (HCV) NS5A open reading frame under the control of the cytomegalovirus immediate early (IE) or a herpes simplex chimeric promoter (alpha 4 gamma 1UL19) were constructed and characterized. Expression studies showed that both HSV-NS5A recombinant viruses were able to express high levels of the NS5A protein in infected cells. Most importantly, using this system, we demonstrated that the NS5A protein interacts with the growth receptor-bound protein 2 (Grb2) and inhibits the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HeLa, NIH3T3 or liver infected cells. Thus, our studies confirm the ability of the NS5A protein to perturb the extracellular signal-regulated kinase (ERK) pathway in HeLa cells by the use of an alternative system for NS5A expression and extend this observation to additional cell lines. We conclude that HSV-based viral vectors may provide a useful system for studying the expression and selected functional properties of the HCV NS5A protein.

Abscisic acid-induced transcription is mediated by phosphorylation of an abscisic acid response element binding factor, TRAB1.

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS-polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain-TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein-TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1.

Differential tissue-specific response to sulfate and methionine of a soybean seed storage protein promoter region in transgenic Arabidopsis.

Expression of the gene encoding the beta subunit of beta-conglycinin, a major soybean seed storage protein, is upregulated by sulfur deficiency and downregulated by methionine (Met). The tissue-specificity of these regulatory mechanisms was studied using a sulfate-responsive region (beta(SR)) from the beta subunit gene promoter. Transgenic Arabidopsis thaliana lines were generated carrying a green fluorescent protein (GFP) reporter gene under control of the cauliflower mosaic virus 35S RNA promoter with a tandem repeat of the beta(SR) element, referred to as the P35S::beta(SR)x3: GFP transgene. Upregulation of P35S::beta(SR)x3:GFP by sulfur deficiency was strongest in leaf margins, where symptoms of sulfur deficiency first appear. P35S::beta(SR)x3:GFP was also upregulated at 2 d after a medium shift from sulfur-sufficient to sulfur-deficient conditions, suggesting that the chimeric promoter is an efficient indicator of sulfur nutritional status. Analysis of transgene expression in a Met-overaccumulating mto1-1 mutation background revealed that the beta(SR) region carries sufficient information for downregulation of promoter activity by Met in developing seeds, but not in young rosettes. Comparisons with another transgenic line, in which the full-length beta promoter is active in non-seed tissues, also suggested that at least two separate tissue-specific mechanisms exist for the downregulation of the beta promoter by Met.

Cell-specific regulatory modules control expression of genes in vascular and visceral smooth muscle tissues.

A novel approach with chimeric SM22alpha/telokin promoters was used to identify gene regulatory modules that are required for regulating the expression of genes in distinct smooth muscle tissues. Conventional deletion or mutation analysis of promoters does not readily distinguish regulatory elements that are required for basal gene expression from those required for expression in specific smooth muscle tissues. In the present study, the mouse telokin gene was isolated, and a 370-bp (-190 to 180) minimal promoter was identified that directs visceral smooth muscle-specific expression in vivo in transgenic mice. The visceral smooth muscle-specific expression of the telokin promoter transgene is in marked contrast to the reported arterial smooth muscle-specific expression of a 536-bp minimal SM22alpha (-475 to 61) promoter transgene. To begin to identify regulatory elements that are responsible for the distinct tissue-specific expression of these promoters, a chimeric promoter in which a 172-bp SM22alpha gene fragment (-288 to -116) was fused to the minimal telokin promoter was generated and characterized. The -288 to -116 SM22alpha gene fragment significantly increased telokin promoter activity in vascular smooth muscle cells in vitro and in vivo. Conversely, a fragment of the telokin promoter (-94 to -49) increased the activity of the SM22alpha promoter in visceral smooth muscle cells of the bladder. Together, these data demonstrate that both vascular- and visceral smooth muscle-specific regulatory modules direct gene expression in subsets of smooth muscle tissues.

Gene regulation of human 12( S)-lipoxygenase

The essential role of the mitogen-activated protein kinase pathway and c- Jun induction in epidermal growth factor (EGF)-induced gene expression of human 12( S)-lipoxygenase was studied. Transfection of cells with Ras, ERK2, Rac and JNK dominant negative mutants all inhibited the EGF-induced promoter activation of the 12( S)-lipoxygenase gene. The EGF induced the expression of c- Jun, and overexpression of c- Jun induced the expression of 12( S)-lipoxygenase mRNA. The Sp1-binding sites in the promoter region of the 12( S)-lipoxygenase gene were requisite for the c- Jun response, which was similar to that observed in the EGF response. Furthermore, the EGF stimulated the interaction between c- Jun and Sp1. The co-expression of the c- Jun dominant negative mutant inhibited the interaction between c- Jun and Sp1, and the promoter activation of the 12( S)-lipoxygenase gene, which was induced by c- Jun overexpression in cells. The EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12( S)-lipoxygenase promoter only when co-expressed with GAL4-c- Jun (1–223) fusion proteins. These results indicate that the direct interaction between c- Jun and Sp1 induced by the EGF cooperatively activated expression of the 12( S)-lipoxygenase gene, and that Sp1 may function at least in part as a carrier to bring c- Jun to the promoter.

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation.

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

An L1 element disrupts human bone sialoprotein promoter: lack of tissue-specific regulation by distalless5 (Dlx5) and runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements

Bone sialoprotein (BSP) is a phosphorylated and sulphated glycoprotein with hydroxyapatite nucleating properties that is specifically expressed in association with physiological and pathological mineralization. Although previous studies have indicated that tissue-specific expression of murine BSP is regulated through a proximal homeodomain element that binds distalless5 (Dlx5) transcription analysis of the homologous human promoter revealed modest enhancement in osteogenic cells. Moreover, whereas forced expression of an antisense Dlx5 vector increased transcription, Dlx5 expression did not alter transcription significantly. Since extended promoter sequences are required to confer absolute tissue-specific expression of BSP in vivo, we characterized the upstream region of the human BSP gene. In contrast to the rat and mouse promoters, which show conserved sequences extending several kbs upstream, analysis of ∼3 kb of the human promoter showed no sequence conservation beyond −0.99 kb. Southern blot analysis of genomic DNA from four different BAC clones showed that this sequence was not an aberration in the human genomic library used to isolate the BSP gene. Using clone BAC H-NH0811I08, the human BSP promoter sequence was extended ∼8 kb upstream from which the non-homologous region was characterized as a 3.48 kb insert coding for an L1 retrotransposon element. Transcriptional analyses of chimeric promoter constructs revealed that the retrotransposon element suppresses transcription <80%. Upstream of the inserted DNA several regions, varying in length from 26 to 161 bps, were conserved within the mouse and human promoters. One of these conserved regions included a runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements consensus element in reverse orientation. Whereas a multimeric form of the element was transcriptionally active in response to Runx2/Cbfa1 when ligated to the BSP basal promoter, the single element in the context of the extended promoter was unresponsive. These studies have characterized the upstream promoter of the human BSP gene, which is interrupted by a unique high-frequency DNA insert that suppresses BSP gene transcription.

Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human β-actin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture.

1368. Transcriptional Targeting of Heterologous Genes to the Liver using Chimeric Promoters: In Vitro and In Vivo Comparative Study

1368. Transcriptional Targeting of Heterologous Genes to the Liver using Chimeric Promoters: In Vitro and In Vivo Comparative Study

1250. Novel Chimeric Gene Promoters Responsive to Hypoxia and Ionizing Radiation for Targeted Gene Therapy of Cancer

1250. Novel Chimeric Gene Promoters Responsive to Hypoxia and Ionizing Radiation for Targeted Gene Therapy of Cancer

1177. Novel Chimeric Prostate-Specific Promoter, PSES-Based Suicide Gene Therapy Demonstrates Specific Killing in Human Prostate Cancer Cells

1177. Novel Chimeric Prostate-Specific Promoter, PSES-Based Suicide Gene Therapy Demonstrates Specific Killing in Human Prostate Cancer Cells

A 235-bp region from a nutritionally regulated soybean seed-specific gene promoter can confer its sulfur and nitrogen response to a constitutive promoter in aerial tissues of Arabidopsis thaliana

The seed-specific promoter of the β subunit gene of β-conglycinin, a major seed storage protein of soybean, is upregulated in response to both sulfur and nitrogen nutrition. This response was studied in non-seed tissues of transgenic Arabidopsis thaliana by fusing the β subunit promoter (Pβ) downstream of an enhancer from the cauliflower mosaic virus 35S RNA promoter (P35S) and using the β-glucuronidase (GUS) reporter gene. The chimeric promoter was upregulated by sulfur deficiency (ΔS), indicating that the Pβ is able to direct the ΔS response in non-seed tissues. Deletion analysis of the Pβ revealed that, in seeds, the −307–−73 bp region is sufficient for the ΔS response. Insertion of the 235 bp region into the P35S conferred the ΔS response on this constitutive promoter in both seed and non-seed tissues. The extent of upregulation by ΔS was more evident when three of the 235 bp fragments were tandemly inserted into the P35S. The findings presented here suggest that a common mechanism exists for sulfur-regulated expression of the Pβ in both seed and non-seed tissues and that this 235 bp region of the Pβ is sufficient for regulation by both sulfur and nitrogen nutrition.

Design of a chimeric promoter induced by pro-inflammatory mediators in articular chondrocytes

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [−247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1β (IL-1β), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1β, and 15-deoxy Δ 12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.

Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice.

The spatial and temporal expression patterns of the rice VP1 (OSVP1) gene, as well as the OSEM gene which it controls, were studied during seed development by in situ hybridization and immuno-localization techniques. The expression of OSVP1 could be detected in embryos as early as 2-3 d after pollination (DAP) and thereafter became preferentially localized to shoot, radicle and vascular tissues during the embryo development at both the mRNA and protein levels. In the aleurone layers, OSVP1 mRNA and protein were detected after 6 DAP. OSEM mRNA was detectable after 6 DAP in the embryo and aleurone tissue. The spatial distribution within the embryo of OSEM mRNA and OSVP1 mRNA/protein was very similar after 6 DAP. Transgenic rice carrying a beta-glucuronidase (GUS) gene transcribed from a chimeric promoter consisting of the CaMV 35S minimal promoter (-46) and the 55-bp promoter fragment of OSEM, minimally required for ABA and VP1 regulation, also exhibited a spatial pattern of GUS expression similar to that of OSEM and OSVP1. These results suggest that (OS)VP1 is a major determinant not only of the seed specificity but also of the spatial pattern of OSEM expression in the developing seed.

Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over-expression in plants

To develop strong promoters for protein over-expression in both dicots and monocots, we constructed a new family of chimeric promoters using sequences of the Commelina Yellow Mosaic Virus (CoYMV), of the Cassava Vein Mosaic Virus (CsVMV) and activating sequences from the CaMV 35S promoter. The chimeric promoters were cloned upstream from the gusA reporter gene. The constructs were used in transient expression experiments, via DNA-coated gold particle delivery to tobacco leaves and maize endosperms. The results showed that candidates among the chimeric promoters could drive expression of the reporter gene to very high levels in the dicot plant tobacco, and all chimeric promoters showed higher expression in maize endosperm than the maize γ-zein promoter used as reference for the monocot expression. Expression cassettes were then used in stable tobacco transformation. Determination of GUS activity throughout growth of the primary transformants showed that two promoters (MPr1163 and MPr1165) could drive expression three to five-fold higher than the highly efficient enhanced 35S promoter. The use of MPr1163 was additionally validated for successful heterologous protein production of human lactoferrin in tobacco via agroinfiltration.

Cbfa1 does not regulate RANKL gene activity in stromal/osteoblastic cells

The rates of osteoblast and osteoclast formation are tightly balanced, possibly due to the requirement of mesenchymal osteoblast progenitors for osteoclastogenesis. Osteoblast differentiation requires the transcription factor Cbfa1, whereas osteoclastogenesis results from the interaction between receptor activator of NFκB ligand (RANKL), expressed on stromal/osteoblastic cells, and RANK, a surface receptor on hematopoietic precursors. A striking decrease in the number of osteoclasts in Cbfa1-deficient mice suggested that Cbfa1 might be involved in RANKL expression. To investigate this possibility and to elucidate the mechanisms regulating RANKL expression, we isolated the 5′-flanking region of the murine RANKL gene and found that it contains two potential binding sites for Cbfa1 (OSE2-like sites). Cbfa1 bound to either of these sites in gel shift assays and stimulated the activity of a chimeric promoter consisting of multimerized RANKL OSE2-like sites inserted upstream from a minimal thymidine kinase (tk) promoter in transient transfections. However, Cbfa1 cotransfection did not stimulate murine RANKL promoter-luciferase constructs. Further analysis revealed that removal of these sites from the RANKL promoter by either site-directed mutagenesis or 5′-deletion did not alter the basal activity of promoter-reporter constructs. Conditional expression of Cbfa1 in a stromal/osteoblastic cell line stimulated osteocalcin mRNA by fivefold, but had no significant effect on RANKL mRNA levels. Conversely, conditional expression of a dominant-negative form of Cbfa1 in the same cell line inhibited osteocalcin mRNA by threefold, but had no effect on RANKL mRNA. Although these results cannot rule out a novel function for Cbfa1 in RANKL expression, they demonstrate that Cbfa1 does not regulate RANKL gene activity in the same manner as known targets of this transcription factor, such as osteocalcin.