Herpes Simplex Virus VP16 Protein Research Articles

Development of a Reporter System Monitoring Regulated Intramembrane Proteolysis of the Transmembrane bZIP Transcription Factor ATF6α.

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of ATF6α by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human ATF6α N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-hATF6α deletion variant was cleaved to liberate active transcription activator encompassing GV-hATF6α fragment which could translocate into the nucleus. The translocated GV-hATF6α fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-hATF6α(333) represents an innovative tool to investigate regulated intramembrane proteolysis of ATF6α. It can substitute active pATF6(N) binding motif-based reporter cell lines.

Construction of enhanced transcriptional activators for improving cellulase production in Trichoderma reesei RUT C30

Enhancing cellulase production in Trichoderma reesei is of great interest for an economical biorefinery. Artificial transcription factors are a potentially powerful molecular strategy for improving cellulase production in T. reesei. In this study, enhanced transcriptional activators XYR1VP, ACE2VP, and ACE1VP were constructed by linking the C terminus of XYR1, ACE2, or ACE1 with an activation domain of herpes simplex virus protein VP16. T. reesei transformants TXYR1VP, TACE2VP, and TACE1VP showed improved cellulase and/or xylanase production. TXYR1VP has a cellulase-free phenotype but with significantly elevated xylanase production. Xylanase I and xylanase II activities [U/(mg biomass)] increased by 51% and 80%, respectively, in TXYR1VP in comparison with parental strain RUT C30. The filter paper activity of TACE2VP in the Avicel-based medium increased by 52% compared to that of RUT C30. In the Avicel-based medium, TACE1VP manifested an 80% increase in FPase activity and a 50% increase in xylanase activity as compared to those of RUT C30. Additionally, when pretreated corn stover was hydrolyzed, crude enzymes produced from TACE1VP yielded a greater glucose release than did the enzymes produced by parental strain RUT C30.

Expression and localization of Luman/CREB3 in mouse embryos during the pre-implantation period.

Luman/CREB3 is a transcription factor that is a member of the cAMP-response-element-binding protein family of basic region-leucine zipper transcription factors. This protein interacts with host cell factor 1, which also associates with the herpes simplex virus protein VP16 to induce the transcription of herpes simplex virus. Currently, the physiological function of Luman/CREB3 in reproductive processes remains unclear. In this study, quantitative real-time PCR and immunofluorescence assays were used to investigate the expression and localization of Luman in mouse oocytes as well as in early embryonic development. Luman protein was detected in the germinal vesicle and metaphase II stage oocytes, and was distributed in the cytoplasm, nucleus, and polar body of the oocyte stage. However, Luman protein and mRNA expression levels were significantly (P < 0.05) increased before activation of the zygotic genome, and expression levels peaked in 4-cell embryos. Expression levels were significantly (P < 0.05) decreased following the 8-cell stage throughout the blastocyst stage. The Luman protein was also distributed in the nucleus and cytoplasm in the early preimplantation embryo and showed enhanced nuclear staining starting from the 2-cell stage embryo up to the 8-cell stage embryo. The differences in the expression and localization of Luman in mouse oocytes and early embryo suggested that Luman plays an important role in oocyte maturation and early embryonic development processes.

Interleukin-6 induces neuroendocrine differentiation (NED) through suppression of RE-1 silencing transcription factor (REST).

Paracrine interleukin-6 (IL-6) can mediate neuroendocrine (NE) features, including the acquisition of a neurite-like phenotype and growth arrest in prostate cancer cells. However, little is known about the mechanisms underlying neuroendocrine differentiation induced by IL-6. Immunoblotting was performed to determine the status of RE1-silencing transcription factor (REST) and of neuroendocrine markers such as Neuron-specific Enolase (NSE), chromogranin A and synaptophysin in LNCaP cells treated with IL-6. To further study the impact of REST-mediated repression on neuroendocrine differentiation (NED) in LNCaP cells, either wild-type REST or a dominant-positive form of REST, REST-VP16, in which both repressor domains of REST were replaced with the activation domain of the herpes simplex virus protein VP16, was introduced into LNCaP cells. In this study, we show that REST is suppressed in IL-6-induced neuroendocrine differentiation in LNCaP cells. Overexpression of exogenous REST abrogated IL-6-induced NED in prostate cancer cells. Expression of the recombinant REST-VP16 fusion protein activated REST target genes and other neuronal differentiation genes and produced neuronal physiological properties. In addition, REST protein turnover was accelerated in IL-6 induced NE differentiated LNCaP cells via the ubiquitin-proteasome pathway, accompanied by a decrease in the expression of the deubiquitylase HAUSP, indicating that pathway(s) priming REST degradation may be involved in IL-6 induced NE differentiation. These results demonstrate that REST functions as a major switch of IL-6 induced neuroendocrine differentiation in LNCaP cells.

Negative Regulation-Resistant p53 Variant Enhances Oncolytic Adenoviral Gene Therapy

Intact p53 function is essential for responsiveness to cancer therapy. However, p53 activity is attenuated by the proto-oncoprotein Mdm2, the adenovirus protein E1B 55kD, and the p53 C-terminal domain. To confer resistance to Mdm2, E1B 55kD, and C-terminal negative regulation, we generated a p53 variant (p53VPΔ30) by deleting the N-terminal and C-terminal regions of wild-type p53 and inserting the transcriptional activation domain of herpes simplex virus VP16 protein. The oncolytic adenovirus vector Ad-mΔ19 expressing p53VPΔ30 (Ad-mΔ19/p53VPΔ30) showed greater cytotoxicity than Ad-mΔ19 expressing wild-type p53 or other p53 variants in human cancer cell lines. We found that Ad-mΔ19/p53VPΔ30 induced apoptosis through accumulation of p53VPΔ30, regardless of endogenous p53 and Mdm2 status. Moreover, Ad-mΔ19/p53VPΔ30 showed a greater antitumor effect and increased survival rates of mice with U343 brain cancer xenografts that expressed wild-type p53 and high Mdm2 levels. To our knowledge, this is the first study reporting a p53 variant modified at the N terminus and C terminus that shows resistance to degradation by Mdm2 and E1B 55kD, as well as negative regulation by the p53 C terminus, without decreased trans-activation activity. Taken together, these results indicate that Ad-mΔ19/p53VPΔ30 shows potential for improving p53-mediated cancer gene therapy.

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.

Anhydrotetracycline–peptide conjugates as representatives for ligand-based transactivating systems

Bioconjugates of anhydrotetracycline and minimal activation sequences (VP1, VP2) derived from the Herpes simplex virus protein VP16 were synthesized. Different ligation strategies were applied and the resulting molecules tested in HeLa cells expressing the reverse transactivator rtTA-S3 for activity. The data clearly demonstrate that the atc-peptide conjugates are able to penetrate the cell membrane. Furthermore, binding to and induction of rtTA-S3 were detected. Structure-activity relationships indicated that the biological activity of the atc-peptide strongly depends on the specific linker used. The N-terminally linked oxime derivative 10 proved excellent activity when the increase of luciferace activity indicated a transcriptional activation substantially exceeding the inducing properties of anhydrotetracycline.

Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems for a beta-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter in transgenic tobacco plants. On treatment with the AhR ligands 3-methylcholanthrene (MC), beta-naphthoflavone (betaNF), and indigo, the transgenic tobacco plants exhibited enhanced GUS activity, presumably by inducible expression of the reporter gene. The recombinant AhR (AhRV), with the activation domain replaced by that of the Herpes simplex virus protein VP16, induced GUS activity much more than the wild-type AhR in the transgenic tobacco plants. Plants carrying AhRV expressed the GUS reporter gene in a dose- and time-dependent manner when treated with MC; GUS activity was detected at 5 nM MC on solid medium and at 12 h after soaking in 25 microM MC. Histochemical GUS staining showed that this system was active mainly in leaf and stem. These results suggest that the AhR-mediated reporter gene expression system has potential for the bioassay of dioxins in the environment and as a novel gene expression system in plants.

Modulation of F1 hybrid stature without altering parent plants through trans‐activated expression of a mutated rice GAI homologue

Hybrid breeding, by taking advantage of heterosis, brings about many superior properties to the F1 progeny. However, some properties, such as increased plant height, are not desirable for agronomic purposes. To specifically counter the height increase associated with hybrid progeny, we employed an Arabidopsis model and tested a trans-activation system for specifically expressing a mutated GAI gene only in the F1 hybrid plants to reduce plant stature. A transcriptional activator, the Gal4 DNA-binding domain fused to the acidic activation domain of herpes simplex virus VP16 protein, driven by a maize ubiquitin promoter, was introduced in one parental line. A rice GAI homologue with an N-terminal deletion of the DELLA domain, driven by a promoter that is responsive to the transcriptional activator, was transferred into another parental line. After genetic crossing, trans-activation of the GAI mutant gene resulted in a dwarf phenotype. Over 50 pair-wise crosses between the parental lines were performed, and analyses suggested that the percentage of F1 progeny exhibiting dwarfism ranged from about 25% to 100%. Furthermore, the dwarfism trait introduced in F1 progeny did not seem to affect total seed yield. Our result suggests the feasibility of manipulating F1 hybrid progeny traits without affecting parent plants or the agronomic property of the progeny.

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

Stabilization but Not the Transcriptional Activity of Herpes Simplex Virus VP16-Induced Complexes Is Evolutionarily Conserved among HCF Family Members

The human herpes simplex virus (HSV) protein VP16 induces formation of a transcriptional regulatory complex with two cellular factors-the POU homeodomain transcription factor Oct-1 and the cell proliferation factor HCF-1-to activate viral immediate-early-gene transcription. Although the cellular role of Oct-1 in transcription is relatively well understood, the cellular role of HCF-1 in cell proliferation is enigmatic. HCF-1 and the related protein HCF-2 form an HCF protein family in humans that is related to a Caenorhabditis elegans homolog called CeHCF. In this study, we show that all three proteins can promote VP16-induced-complex formation, indicating that VP16 targets a highly conserved function of HCF proteins. The resulting VP16-induced complexes, however, display different transcriptional activities. In contrast to HCF-1 and CeHCF, HCF-2 fails to support VP16 activation of transcription effectively. These results suggest that, along with HCF-1, HCF-2 could have a role, albeit probably a different role, in HSV infection. CeHCF can mimic HCF-1 for both association with viral and cellular proteins and transcriptional activation, suggesting that the function(s) of HCF-1 targeted by VP16 has been highly conserved throughout metazoan evolution.

Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of genes involved in O(2) homeostasis, including vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis. We sought to exploit this native adaptive response to hypoxia as a treatment for chronic ischemia. A hybrid protein consisting of DNA-binding and dimerization domains from the HIF-1alpha subunit and the transactivation domain from herpes simplex virus VP16 protein was constructed to create a strong, constitutive transcriptional activator. After transfection into HeLa, C6, and Hep3B cells, this chimeric transcription factor was shown to activate expression of the endogenous VEGF gene, as well as several other HIF-1 target genes in vitro. The bioactivity of HIF-1alpha/VP16 hybrid gene transfer in vivo was examined in a rabbit model of hindlimb ischemia. Administration of HIF-1alpha/VP16 was associated with significant improvements in calf blood pressure ratio, angiographic score, resting and maximal regional blood flow, and capillary density (all P:<0.01). The HIF-1alpha/VP16 hybrid transcription factor is able to promote significant improvement in perfusion of an ischemic limb. These results confirm the feasibility of a novel approach for therapeutic angiogenesis in which neovascularization may be achieved indirectly by use of a transcriptional regulatory strategy.

Functional Independence of the Two Cysteine-rich Activation Domains in the Yeast Mac1 Transcription Factor

Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the (264)CXC[(X)(4)]CXC[(X)(2)]C[(X)(2)][H(279)]C1 motif. The lone exception was a L260S substitution. Two additional MAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain.

The Splicing Factor U1C Represses EWS/FLI-mediated Transactivation

EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations. In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1. EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor. Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.

Target Selection by Selective Binding

Homeodomain proteins, such as Fushi tarazu (Ftz) are transcription factors that are major regulators of pattern formation early in development; however, homeodomain proteins alone have a low affinity and low specificity for DNA. Thus, their ability to regulate target gene activity is dependent on their interaction with cofactors. Two models for how these cofactors regulate homeodomain proteins are that the cofactors bind DNA specifically and direct the homeodomain proteins to these specific sites (selective binding model) or that the homeodomain proteins bind to many genes, and the cofactors act to control the transcriptional activity of the homeodomain proteins at specific sites (selective activation model). Nasiadka et al . replaced the activation domain of Ftz with the strong transcriptional activation domain of the herpes simplex virus protein VP16 and expressed this fusion protein in developing fly embryos to test the two models of regulation. The authors found that the FtzVP16 protein did not result in activation of genes not normally regulated by Ftz, suggesting that Ftz binding was controlling gene selection not selective activation. Two additional pieces of data in support of the selective binding model were that the phenotypes resulting from expression of FtzVP16 were predominantly the same as those caused by misexpression of Ftz and that homeodomain-deleted versions of Ftz and FtzVP16 were able to regulate Ftz target genes. Nasiadka, A., Grill, A., and Krause, H.M. (2000) Mechanisms regulating target gene selection by the homeodomain-containing protein Fushi tarazu. Development 127 : 2965-2976. [Online Journal]

Nuclear endpoint of Wnt signaling: neoplastic transformation induced by transactivating lymphoid-enhancing factor 1.

The interaction between beta-catenin and LEF-1/TCF transcription factors plays a pivotal role in the Wnt-1 signaling pathway. The level of beta-catenin is regulated by partner proteins, including glycogen synthase kinase-3beta (GSK-3beta) and the adenomatous polyposis coli (APC) tumor suppressor protein. Genetic defects in APC are responsible for a heritable predisposition to colon cancer. APC protein and GSK-3beta bind beta-catenin, retain it in the cytoplasm, and facilitate the proteolytic degradation of beta-catenin. Abrogation of this negative regulation allows beta-catenin to translocate to the nucleus and to form a transcriptional activator complex with the DNA-binding protein lymphoid-enhancing factor 1 (LEF-1). This complex is thought to be involved in tumorigenesis. Here we show that covalent linkage of LEF-1 to beta-catenin and to transcriptional activation domains derived from the estrogen receptor or the herpes simplex virus protein VP16 generates transcriptional regulators that induce oncogenic transformation of chicken embryo fibroblasts. The chimeras between LEF-1 and beta-catenin or VP16 are constitutively active, whereas fusions of LEF-1 to the estrogen receptor are regulatable by estrogen. These experiments document the oncogenicity of transactivating LEF-1 and show that the transactivation domain normally provided by beta-catenin can be replaced by heterologous activation domains. These results suggest that the transactivating function of the LEF-1/beta-catenin complex is critical for tumorigenesis and that this complex transforms cells by activating specific LEF-1 target genes.

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal.

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

The activities of acidic and glutamine-rich transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression.

The aim of this work was to design strong transcriptional activators that can be used to regulate plant gene expression. The contribution of different components in a transcription factor and target gene system was assayed by measuring transcriptional activation. Each component was optimised to achieve maximal reporter gene expression in transient protoplast transformation assays. The DNA-binding domain of the yeast transcriptional activator GAL4 was studied in the context of fusion proteins with activation domains of the herpes simplex virus protein VP16 or the tomato Myb-like activator THM18. Multimerisation of the activation domain and insertion of a homopolymeric glutamine stretch was used to increase transcription factor potency. Evidence is presented that these modifications can result in even more active transcription factors when they are combined. Finally, it was demonstrated using competition experiments that transcription factors with acidic activation domains can mutually suppress their activation potentials when expressed at high levels.

Viral mimicry: common mode of association with HCF by VP16 and the cellular protein LZIP.

Upon infection of human cells, the herpes simplex virus protein VP16 associates with the endogenous cell-proliferation factor HCF. VP16 can also associate with HCFs from invertebrates, suggesting that VP16 mimics a cellular protein whose interaction with HCF has been conserved. Here, we show that VP16 mimics the human basic leucine-zipper protein LZIP, which, through association with HCF, may control cell-cycle progression. VP16 and LZIP share a tetrapeptide motif-D/EHXY-used to associate with human HCF. The LZIP-related Drosophila protein BBF-2/dCREB-A contains this HCF-binding motif, indicating that the LZIP-HCF interaction has been conserved during metazoan evolution.