Activate Reporter Gene Expression Research Articles

SKIP modifies gene expression by affecting both transcription and splicing

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a β-thalassemia mutation in the human β-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.

Transcriptional targeting in ovarian cancer cells using the human epididymis protein 4 promoter

Objective. Limitations of current ovarian cancer gene therapies include lack of specificity and transduction of normal tissues. One strategy toward overcoming these limitations is to direct gene therapy specifically to ovarian cancer cells by using tissue- and tumor-specific promoters. The whey-acidic protein human epididymis protein 4 (HE4) is frequently overexpressed in ovarian cancer, suggesting that the HE4 promoter is highly transcriptionally active in the disease. The objective of this study was to isolate the HE4 promoter and examine its ability to selectively activate reporter gene expression in an ovarian cancer-specific manner. Methods. To investigate transcriptional targeting in ovarian cancer gene therapy, we isolated a region of the HE4 promoter from −530 to +122 (pHE4-652; relative to the ATG start site of HE4) and placed it upstream of a luciferase reporter gene plasmid to generate pHE4-652-luc. The activity of the pHE4-652-luc reporter construct was characterized in transient transfection assays in a panel of epithelial ovarian cancer cell lines (SKOV-3, SKOV-3×, CP70, HeyC2, A2780, A2780CP, OVCAR-3), non-ovarian tumor cell lines, and primary cultures of normal cells. The activity of two other candidate gene therapy promoters, human telomerase reverse transcriptase ( hTERT) and OSP1, was also characterized in these cell lines. Results. The HE4 promoter was active in 5/7 ovarian cancer cell lines with the range of activity spanning 0.06- to 3-fold that observed for a positive control, cotransfected reporter construct (SV-40-luc). Minimal pHE4-652 promoter activity, defined as ≤5% of the activity detected with the SV-40-luc construct, was observed in the non-ovarian tumor cell lines and normal cells. The hTERT and the OSP1 promoters were active in the ovarian cancer lines. hTERT activity was highest in the CP70 cell line, and OSP1 activity was highest in the SKOV-3× cell line. Modest OSP1 and hTERT promoter activity was observed in normal cell lines and in selected non-ovarian cancer cell lines. Conclusion. This is the first report using the pHE4-652 promoter to drive specific reporter gene expression in epithelial ovarian cancer cell lines, and we are continuing to develop this promoter for use in transcriptional targeting in ovarian cancer gene therapy.

Control of gene expression with small molecules: biotin-mediated acylation of targeted lysine residues in recombinant yeast.

Chemical inducers of dimerization (CIDs) are powerful tools for controlling diverse cellular processes. These small molecules typically form strong noncovalent interactions with proteins. We report a related approach involving covalent acylation of a specific lysine residue of a target protein by the small molecule biotin. To control protein-protein interactions with biotin, the biotin protein ligase BirA from E. coli was coexpressed in yeast with a streptavidin-LexA fusion protein and Avitag or BCCP biotin acceptor peptides fused to the B42 activation domain. The addition of biotin (10 nM) resulted in BirA-mediated biotinylation of the biotin acceptor protein, recruitment to LexA DNA sites, and maximal activation of reporter gene expression in this yeast tribrid system. The high potency, low toxicity, and low molecular weight of biotin as a covalent CID are attractive properties for controlling cellular processes.

Selective modulation of the SM22alpha promoter by the binding of BTEB3 (basal transcription element-binding protein 3) to TGGG repeats.

We have previously identified a C2H2 zinc-finger transcription factor [BTEB3 (basal transcription element-binding protein 3)/KLF13 (Krüppel-like factor 13)] that activates the minimal promoter for the smooth muscle-specific SM22alpha gene in other types of cell. We show that recombinant BTEB3 binds to three TGGG motifs in the minimal SM22alpha promoter. By mutation analysis, only one of these boxes is required for BTEB3-dependent promoter activation in P19 cells and BTEB3 activates or inhibits reporter gene expression depending on the TGGG box to which it binds. Transient transfection experiments show that BTEB3 also activates reporter gene expression from the SM22alpha promoter in VSMCs (vascular smooth muscle cells). Similar studies showed that BTEB3 did not activate expression from the promoter regions of the smooth muscle myosin heavy chain or smooth muscle alpha-actin promoters, which contain similar sequences, implying that promoter activation by BTEB3 is selective. The expression of BTEB3 is readily detectable in VSMCs in vitro and is modulated in response to injury in vivo.

Synthesis of a beta-estradiol-biotin chimera that potently heterodimerizes estrogen receptor and streptavidin proteins in a yeast three-hybrid system.

Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.

Isolation and characterization of the human CP49 gene promoter.

This study focuses on the identification of regulatory elements that contribute to lens-specific expression of the human CP49 gene within the 5'-flanking DNA sequences. The DNA sequence upstream of the human CP49 coding region was subcloned as a set of 5' and 3' deletion series. The constructs were transfected into lens (N/N1003A) and nonlens (NIH3T3) cell lines and chicken primary lens cultures, to test for promoter activity and specificity. To further test the specificity, a portion of the 5' flanking DNA sequence was used to drive transgene expression in mice. The flanking DNA sequence was analyzed for potential transcription factor-binding sites. The 5'-flanking DNA preferentially activated reporter gene expression in a lens-preferred manner when transfected into cultured cells. Transgene expression driven by the CP49 promoter region was lens specific. Analysis of the proximal promoter sequence revealed the presence of potential binding sites for the AP-1, AP-2, and OCT-1 transcription factors and the absence of TATA and CAAT boxes. The sequence upstream of the CP49 gene possesses promoter activity and is able to drive lens-preferred expression in both transfection and transgenic experiments. Promoter activity is dependent on the presence of the proximal 300 bp directly upstream of the coding region.

Mammalian metal response element-binding transcription factor-1 functions as a zinc sensor in yeast, but not as a sensor of cadmium or oxidative stress.

The zinc finger protein, metal response element-binding transcription factor-1 (MTF-1) regulates the expression of genes in response to metal ions and oxidative stress. The precise mechanisms by which this occurs are not understood. To further examine this problem, mouse MTF-1 was expressed in Saccharomyces cerevisiae and tested for the ability to activate metal response element-driven reporter gene expression. Zinc was an effective inducer of reporter gene expression. In general, the magnitude of zinc induction was dependent on the concentration of zinc in the culture medium, but independent of the amount of MTF-1 expression. Zinc induction also occurred with either integrated or episomal reporter plasmids containing the native mouse metallothionein-I proximal promoter. Deletion of fingers 5 and 6 of MTF-1, which function in a zinc-dependent manner to stabilize the DNA-binding activity of the protein in vitro, did not diminish the zinc induction of either episomal or integrated promoters. However, a Gal4 DNA-binding domain- MTF-1 fusion protein, which binds constitutively to the Gal4-responsive promoter, was not zinc inducible but caused constitutive activation of reporter gene expression. This suggests that zinc activation of the DNA-binding activity of MTF-1 is the rate limiting step in its metalloregulatory function in yeast. In contrast, MTF-1 was not responsive to either cadmium or hydrogen peroxide, suggesting that distinct co-activators or signal transduction cascades not found in yeast are required to mediate MTF-1 activation of gene expression by this toxic metal and by oxidative stress.

Expression of the Human Poliovirus Receptor/CD155 Gene Is Activated by Sonic Hedgehog

The human poliovirus receptor/CD155 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. The ectodomain of CD155 mediates cell attachment to the extracellular matrix molecule vitronectin, while its intracellular domain interacts with the dynein light chain Tctex-1. CD155 is a primate-restricted gene that is expressed during development in mesenchymal tissues and ventrally derived structures within the CNS. Its function in adults is as yet unknown, but significantly, CD155 is aberrantly expressed in neuroectodermal tumors. We show that the expression of CD155 mRNA is up-regulated when human Ntera2 cells are treated with purified Sonic hedgehog (Shh) protein. Reporter gene expression driven by the CD155 core promoter is activated by Shh in transient co-transfection assays. Analysis of the CD155 core promoter indicates that an intact GLI binding site is required for Shh activation. In addition, overexpression of Gli1 or Gli3 potently activates reporter gene expression driven by the CD155 core promoter. These data identify the CD155 gene as a transcriptional target of Shh, a finding that has significance for the normal function of CD155 during development and the expression of CD155 in neuroectodermal tumors.

A Combination of cis-Acting Elements Is Required to Activate the Pro-α1(I) Collagen Promoter in Tendon Fibroblasts of Transgenic Mice

The genes encoding the two type I collagen chains are selectively activated in few cell types, including fibroblasts and osteoblasts. By generating transgenic mice, we have previously shown that the activity of the mouse pro-alpha1(I) promoter was controlled by separate cell-specific cis-acting elements. In particular, a sequence located between -3.2 and -2.3 kb was needed to induce expression of the reporter gene at high levels in tendon fibroblasts. In the present work, by using the same transgenic approach, we have identified two short elements in this sequence, named tendon-specific element (TSE) 1 and TSE2, that were necessary to direct reporter gene expression selectively in tendon fibroblasts. Gel shift assays showed that TSE1 and TSE2 bound proteins specifically present in nuclear extracts from tendon fibroblasts and that the sequence of TSE2 binding a tendon-specific protein corresponded to an E-box. Analysis of transgenic mice further indicated that TSE1 and TSE2 needed to cooperate not only with each other but also with other cis-acting elements of the proximal promoter to activate reporter gene expression in tendon fibroblasts. Similarly, it pointed out that the so-called osteoblast-specific element had to interact with downstream sequences to drive reporter gene expression in osteoblasts of transgenic mice. Thus, expression of the mouse pro-alpha1(I) collagen gene in tendon fibroblasts appears to be the result of a unique combination of different cis-acting elements, including TSE1 and TSE2.

Induction of CYP3A expression by dehydroepiandrosterone: involvement of the pregnane X receptor.

Dehydroepiandrosterone (DHEA) is a steroid produced by the human adrenal gland. Administration of pharmacological doses of DHEA to rats changes expression of many genes, including the cytochrome P450 family members CYP4A1 and CYP3A23. It is known that induction of CYP4A expression by DHEA requires the peroxisome proliferator-activated receptor alpha (PPAR(alpha)). In the current study, PPAR(alpha)-null mice were used to examine the role of PPAR(alpha) in expression of CYP3A. In wild-type mice, 150 mg/kg DHEA-sulfate induced Cyp4a and Cyp3a11 mRNAs by 5- and 2-fold, respectively. Induction of Cyp4a expression by DHEA-sulfate was not observed in PPAR(alpha)-null mice, whereas induction of Cyp3a11 expression by DHEA-sulfate was similar between genotypes. This suggests that PPAR(alpha) is not involved in induction of Cyp3a11 expression by DHEA. Because expression of CYP3A family members can be induced by activation of another member of the nuclear receptor superfamily, the pregnane X receptor (PXR), we examined the ability of DHEA to activate PXR. In transient transfection assays, DHEA and its metabolites androst-5-ene-3beta,17beta-diol (ADIOL), androst-5-ene-3,17-dione, and androst-4-ene-3,17-dione were activators of PXR. Maximal induction of a PXR-responsive reporter gene of approximately 3-fold was observed at concentrations of 50 to 100 microM, indicating that these steroids are relatively weak activators of PXR. Human and murine PXR exhibited different specificities for DHEA and its metabolites. ADIOL activated reporter gene expression in the presence of murine but not human PXR. Results of these studies suggest that the induction of rodent CYP3A expression upon treatment with high doses of DHEA occurs through activation of PXR.

Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis. Functional analysis of the promoter identifies a calcium sensitive region required for basal activity

To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5′ region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5′ end of the xMGP promoter revealed a minimal activating element in the sequence from −70 to −36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from −180 to −36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from −70 to −36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression.

Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis. Functional analysis of the promoter identifies a calcium sensitive region required for basal activity.

To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5' region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5' end of the xMGP promoter revealed a minimal activating element in the sequence from -70 to -36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from -180 to -36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from -70 to -36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression.

A GAL4-based yeast three-hybrid system for the identification of small molecule–target protein interactions

We report the development of a yeast strain designed for assaying compound–protein interactions through activation of reporter gene expression. Activation of lacZ expression, driven by the GAL4 promoter, has been demonstrated for precedented compound–protein interactions between FK506 and FK506 binding protein 12 (FKBP12) and also between methotrexate (MTX) and dihydrofolate reductase (DHFR). Reporter gene expression was completely abrogated in a competitive manner by the presence of excess FK506 or MTX, respectively. In addition, a strain expressing a mutated DHFR clone with decreased binding affinity for MTX was not capable of activating reporter gene expression. While strain sensitivity is compound-dependent, the minimum compound concentration necessary to drive reporter gene expression was 20nM for the FK506–FKBP12 interaction. The utility of this strain as a tool for identifying unknown compound-binding proteins has been demonstrated by screening a mouse cDNA library for clones that encode proteins capable of binding MTX. Four library clones of mouse DHFR were identified after screening 5×106 clones. The screen background was low and false positives were easily identified, making this yeast system particularly amenable for use in a screening context for novel compound–protein interactions.

Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants.

In order to identify genes that are involved in the defense reaction against pathogen attack, we screened for examples that are regulated during the hypersensitive response (HR) to infection by tobacco mosaic virus (TMV) of tobacco ( Nicotiana tabacum cv. Xanthi nc) carrying the N gene, which confers resistance to TMV. Among seven genes initially identified by fluorescent differential display, one clone was further characterized because its transcripts accumulated rapidly and transiently after the onset of HR. Its full-length cDNA of 1346 bp encoded a polypeptide consisting of 258 amino acids. The deduced protein contained a single WRKY domain, a Cys(2)His(2) zinc-finger motif and a leucine-zipper motif, showing high similarity to WIZZ, a member of the family of WRKY transcription factors in tobacco. The gene was thus designated TIZZ. A GFP-TIZZ fusion protein was found to localize to the nucleus upon introduction into epidermal cells of onion. Bacterially expressed TIZZ was able to bind to the W-box (TTGAC) element that is recognized by other WRKY proteins, but transactivation assays showed it to be unable to activate reporter gene expression by itself. TIZZ transcripts were induced in TMV-infected nahG transgenic tobacco plants, in which salicylic acid fails to accumulate. Neither exogenously applied salicylic acid nor mechanical wounding induced TIZZ transcript accumulation. These results indicate the presence of salicylic acid-independent pathways for HR signal transduction, in which a novel type of WRKY protein(s) may play a critical role for the activation of defense.

Cre/loxP recombination-activated neuronal markers in mouse neocortex and hippocampus.

A new strategy for visualizing neuronal cell morphology of mouse brain based on Cre/loxP recombination-activated gene expression is described. A "reporter" transgenic line was generated which expressed a fusion gene encoding a dendrite-targeted green fluorescent protein (MAP2-GFP) upon deletion of a transcription/translation STOP (transcription and translation stop signal) cassette. Cre transgenic "deleter" lines were established that activated reporter gene expression at various frequencies in pyramidal neurons in the forebrain. A deleter line was identified which activated a MAP2-GFP reporter gene at very low frequency (less than 0.1% of pyramidal neurons) and allowed the visualization of dendritic structures of individual neocortical and hippocampal pyramidal neurons. In addition, vertical "columns" of pyramidal neurons in the neocortex were labeled in these mice. In a second deleter line, a MAP2-GFP reporter gene was selectively activated in pyramidal neurons of the CA-1 subregion of the hippocampus in young mice. With its combinatorial property, this binary recombination-activated neuronal marker system should facilitate the study of detailed morphology, connectivity, and plasticity of defined classes of live neurons in vitro and in vivo.

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.

The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

Estrogen response element binding induces alterations in estrogen receptor-alpha conformation as revealed by susceptibility to partial proteolysis.

Genes whose expression is highly induced by estradiol (E(2)) contain multiple estrogen response elements (EREs) in their promoters. Previously we reported that estrogen receptor-alpha (ERalpha) binds cooperatively to and E(2) synergistically activates reporter gene expression from three or four tandem copies of a consensus ERE (EREc38). Here we evaluated how ERalpha binding to one, two, three or four tandem copies of EREc38 affects ERalpha conformation as detected by altered ERalpha trypsin digestion patterns in Western blots. E(2)- or 4hydroxytamoxifen (4-OHT)-occupied ERalpha bound to the pS2 ERE or to a single copy of EREc38 showed enhanced susceptibility to trypsin digestion compared to E(2)- or 4-OHT-ERalpha incubated with DNA lacking an ERE. ERalpha binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ERalpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERalpha and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERalpha conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.

A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.

Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription.

Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes. The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors. To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2. We found that Nrf2 contains two transcription activation domains, Neh4 and Neh5, which act synergistically to attain maximum a activation of reporter gene expression. Neh4 and Neh5 both individually and cooperatively bind to CBP (CREB (cAMP Responsive Element Binding protein) Binding Protein). In fact, the specific inhibitor of CBP, adenovirus E1A protein, significantly reduced Nrf2 activity. Importantly, the CBP-binding activity of Nrf2 deletion mutants positively correlated with their transactivation activity. Neh5 contains a motif which is commonly conserved among the CNC factors, whereas Neh4 contains the novel CBP-interacting motif recently identified in p53 and E2F. Our results indicate that Nrf2 exploits the cooperative binding of two independent transactivation domains to CBP in the acquisition of a potent transactivation activity.

Tandem repetition of baculovirus ie1 promoter results in upregulation of transcription.

In order to improve a virus-independent gene expression system in insect cells, the effect of the repetition of the promoter sequence of a baculovirus immediate early gene, BmNPV ie1, on the gene expression activity was examined. Tandem repetition of ie1 promoter resulted in about a 4-fold increase of the reporter gene expression activity. Primer extension analysis revealed that the repetition increased not only the amount of transcripts (4 to 5-times) but also the number of sites of transcription initiation (at least 3 extra initiation sites). A mutational experiment demonstrated the involvement of independent cis-acting elements in these two different promoter-enhancing effects.