Acidic Transcriptional Activation Domain Research Articles

Targeting the targets of Type III effector proteins secreted by phytopathogenic bacteria

Targeting the targets of Type III effector proteins secreted by phytopathogenic bacteria

The virulence factor AvrXa7 of Xanthomonas oryzae pv. oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein.

AvrXa7 is a member of the avrBs3 avirulence gene family, which encodes proteins targeted to plant cells by a type III secretion apparatus. AvrXa7, the product of avrXa7, is also a virulence factor in strain PXO86 of Xanthomonas oryzae pv. oryzae. Avirulence and virulence specificities are associated with the central repeat domain, which, in avrXa7, consists of 25.5 direct repeat units. Mutations in three C-terminal nuclear localization signal motifs eliminated avirulence and virulence activities in rice and severely reduced nuclear localization in a yeast assay system. Both pathogenicity functions and nuclear localization were restored on the addition of the sequence for the nuclear localization signal motif from SV40 T-antigen. The loss of avirulence activity because of mutations in the acidic transcriptional activation domain was restored by addition of the activation domain from the herpes simplex viral protein VP16. The activation domain was also required for virulence activity. However, the VP16 domain could not substitute for the endogenous domain in virulence assays. In gel shift assays, AvrXa7 bound double-stranded DNA with a preference for dA/dT rich sequences. The results indicate that products of the avrBs3-related genes are virulence factors targeted to host cell nuclei and have the potential to interact with the host DNA and transcriptional machinery as part of their mode of action. The results also suggest that the host defensive recognition mechanisms are targeted to the virulence factor site of action.

Truncation of the C-terminal acidic transcriptional activation domain of herpes simplex virus VP16 renders expression of the immediate-early genes almost entirely dependent on ICP0.

The herpes simplex virus (HSV) proteins VP16 and ICP0 play key roles in stimulating the onset of the viral lytic cycle. We sought to explore the regulatory links between these proteins by studying the phenotypes of viral mutants in which the activation functions of both were simultaneously inactivated. This analysis unexpectedly revealed that truncation of the C-terminal transcriptional activation domain of VP16 (allele V422) in an ICP0-deficient background almost completely eliminated immediate-early gene expression and virus replication in Vero and HEL cells. The doubly mutant viral genome persisted in a quiescent state for at least 10 days in HEL cells infected at high multiplicity and could be reactivated by superinfection with wild-type HSV. In contrast, the in1814 VP16 mutation produced a markedly less severe phenotype in the same ICP0-deficient background. These data demonstrate that expression of the immediate-early genes requires ICP0 when the C-terminal activation domain of VP16 is deleted and raise the possibility that the in1814 form of VP16 retains a residual ability to stimulate gene expression during virus infection.

Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1.

Potent induction of the gene coding for human prointerleukin 1beta (il1b) normally requires a far-upstream inducible enhancer in addition to a minimal promoter located between positions -131 and +12. The transcription factor Spi-1 (also called PU.1) is necessary for expression and binds to the minimal promoter, thus providing an essential transcription activation domain (TAD). In contrast, infection by human cytomegalovirus (HCMV) can strongly activate il1b via the expression of immediate early (IE) viral proteins and eliminates the requirement for the upstream enhancer. Spi-1 has been circumstantially implicated as a host factor in this process. We report here the molecular basis for the direct involvement of Spi-1 in HCMV activation of il1b. Transfection of Spi-1-deficient HeLa cells demonstrated both the requirement of Spi-1 for IE activity and the need for a shorter promoter (-59 to +12) than that required in the absence of IE proteins. Furthermore, in contrast to normal, enhancer-dependent il1b expression, which absolutely requires both the Spi-1 winged helix-turn-helix (wHTH) DNA-binding domain and the majority of the Spi-1 TAD, il1b expression in the presence of IE proteins does not require the Spi-1 TAD, which plays a synergistic role. In addition, we demonstrate that a single IE protein, IE2, is critical for the induction of il1b. Protein-protein interaction experiments revealed that the wing motif within the Spi-1 wHTH domain directly recruits IE2. In turn, IE2 physically associates with the Spi-1 wing and requires the integrity of at least one region of IE2. Functional analysis demonstrates that both this region and a carboxy-terminal acidic TAD are required for IE2 function. Therefore, we propose a protein-tethered transactivation mechanism in which the il1b promoter-bound Spi-1 wHTH tethers IE2, which provides a TAD, resulting in the transactivation of il1b.

The C terminus of AvrXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus.

The avirulence gene avrXa10 of Xanthomonas oryzae pv oryzae directs the elicitation of resistance in a gene-for-gene manner in rice lines carrying the resistance gene Xa10. We have localized a transcriptional activator domain in the C terminus of AvrXa10 by using amino acid replacement mutagenesis. One mutant, with replacements at three hydrophobic amino acid residues in the C-terminal domain, was defective for transcriptional activation in yeast and avirulence activity in rice. The activation domain from the herpes virus protein VP16 restored the ability of the bacteria expressing the hybrid protein to elicit a resistance reaction. Elicitation was specific for Xa10, and the reaction had the hallmarks of the response to AvrXa10. The results indicate that a domain with the properties of a transcriptional activator plays a critical role in AvrXa10 function. The results also indicate that the protein has the potential to interact with the plant transcriptional program, although a role for the domain in the stability or conformation of the protein in the plant cannot be excluded. In a broader sense, the transcriptional activation domain of avrXa10 may represent a prokaryotic version of the acidic transcriptional activation domain, which heretofore has been found exclusively in eukaryotes.

Chromatin remodeling and activation of chromosomal DNA replication by an acidic transcriptional activation domain from BRCA1.

An increasing number of transcription factors have been shown to activate DNA replication. However, the underlying mechanism remains to be elucidated. Here it is shown that when tethered to a cellular replication origin, the acidic transcriptional activation domain of the breast cancer protein BRCA1 alters the local chromatin structure and stimulates chromosomal DNA replication. Cancer-predisposing mutations in BRCA1 that abolish transcriptional activation also prevent chromatin remodeling and activation of replication. Chromatin remodeling occurs even in the absence of a functional replication origin. Thus, increasing chromatin accessibility may be an important mechanism used by transcription factors to facilitate multiple nuclear processes.

Viruses, microorganisms and scientists meet the nuclear pore. Leysin, VD, Switzerland, February 26-March 1, 1998.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

The MHC class II transactivator (CIITA) requires conserved leucine charged domains for interactions with the conserved W box promoter element.

The class II transactivator CIITA is required for transcriptional activation of the major histocompatibility complex (MHC) class II genes. Aside from an N-terminal acidic transcriptional activation domain, little is known about how this factor functions. Extensive mutagenesis of CIITA was undertaken to identify structural motifs required for function. The ability of mutants to activate a reporter gene under the control of MHC class II conserved W-X-Y or X-Y regulatory elements was determined. Two mutants displayed differential activity between the two promoters, activating transcription with the W-X-Y but not the X-Y elements. All mutants were tested for their ability to interfere with wild-type CIITA activity. Five CIITA mutant constructions were able to down-regulate wild-type CIITA activity. Three of these mutants contained targeted disruptions of potential functional motifs: the acidic activation domain, a putative GTP-binding motif and two leucine charged domains (LCD motifs). The other two contained mutations in regions that do not have homology to described proteins. The characterization of CIITA mutants that are able to discriminate between promoters with or without the W box strongly suggests that CIITA requires such interactions for function. The identification of LCD motifs required for CIITA function brings to light a previously undefined role of these motifs in CIITA function.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor.

The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.

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.

Truncation of the C-terminal acidic transcriptional activation domain of herpes simplex virus VP16 produces a phenotype similar to that of the in1814 linker insertion mutation.

We examined the phenotype of a herpes simplex virus (HSV) type 1 mutant (V422) in which the C-terminal acidic activation domain of the virion transactivator VP16 is truncated at residue 422. The efficiency of plaque formation by V422 on Vero cells was boosted by approximately 100-fold by including hexamethylene bis-acetimide (HMBA) in the growth medium, as previously observed with the in1814 VP16 linker insertion mutant isolated by Preston and colleagues. V422 displayed severely reduced levels of the immediate-early transcripts encoding ICP0 and ICP4 during infection in the presence of cycloheximide, and this defect was partially overcome by the addition of HMBA. The defect in plaque formation exhibited by V422 and in 1814 was efficiently complemented in U2OS osteosarcoma cells, which had previously been shown to complement ICP0 null mutations. Taken in combination, these data confirm the key role of VP16 in triggering the onset of the HSV lytic cycle.

Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5.

Two of the genes defective in the five complementation groups identified in the class II-negative bare lymphocyte syndrome or corresponding laboratory mutants have been cloned. One gene encodes a protein, RFX5, that is a member of the RFX family of DNA binding proteins. The other, CIITA, encodes a large protein with a defined acidic transcriptional activation domain; this protein does not interact with DNA. Expression plasmids encoding regions of RFX5 fused to the GAL4 DNA binding domain activated transcription from a reporter construct containing GAL4 sites in a cotransfection assay in the Raji human B cell line. However, these plasmids produced transcriptional activity in HeLa cells only in conjunction with interferon gamma stimulation, a condition in which expression of both CIITA and class II major histocompatibility complex surface proteins are induced. Furthermore, these plasmids were not active in RJ2.2.5, an in vitro mutagenized derivative of Raji in which both copies of CIITA are defective. Transcriptional activation by the RFX5 fusion protein could be restored in RJ2.2.5 by cotransfection with a CIITA expression plasmid. Finally, a direct interaction between RFX5 and CIITA was detected with the yeast two-hybrid and far-Western blot assays. Thus, RFX5 can activate transcription only in cooperation with CIITA. RFX5 and CIITA associate to form a complex capable of activating transcription from class II major histocompatibility complex promoters. In this complex, promoter specificity is determined by the DNA binding domain of RFX5 and the general transcription apparatus is recruited by the acidic activation domain of CIITA.

Interaction between acidic transcriptional activation domains of herpes simplex virus activator protein VP16 and transcriptional initiation factor IID

Publisher Summary This chapter discusses the interaction between acidic transcriptional activation domains of herpes simplex virus activator protein VP16 and transcriptional initiation factor IID (TFIID). The chapter describes the use of protein affinity chromatography to identify interactions between acidic activation domains and TFIID. This chapter describes most of the technical details involved in using protein affinity chromatography to probe the structure of multiprotein complexes. The chapter focuses primarily on those technical considerations that are unique for RNA polymerase II and its interacting factors. Of major importance is the biochemical and genetic criteria that are useful for evaluating whether an observed interaction is likely to be specific and biologically important. Evidence suggests that transcription in vivo is carried out by RNA polymerase II holoenzyme containing most or the entire general initiation factors, as well as many other polypeptides. The interaction of activators with any one of these polypeptides in RNA polymerase II holoenzyme may recruit RNA polymerase II to the promoter and lead to transcriptional activation.

Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize.

The Viviparous-1 (VP1) transcriptional activator of maize is required for abscisic acid induction of maturation-specific genes late in seed development leading to acquisition of desiccation tolerance and arrest in embryo growth. Here, we show that VP1 also inhibits induction of the germination-specific alpha-amylase genes in aleurone cells of the developing seed and thereby appears to be involved in preventing precocious hydrolyzation of storage compounds accumulating in the endosperm. In developing seeds of the somatically instable vp1-m2 mutant, hydrolase activity was derepressed specifically in endosperm sectors underlying vp1 mutant aleurone. A barley alpha-amylase promoter-GUS reporter construct (Amy-GUS) was induced in developing vp1 mutant aleurone cells but not in wild-type aleurone cells. Moreover, transient expression of recombinant VP1 and vp1 mutant aleurone cells strongly inhibited expression of Amy-GUS and thus effectively complemented this aspect of the mutant phenotype. VP1 specifically repressed induction of Amy-GUS by the hormone gibberellic acid in aleurone of germinating barley seeds. Deletion of the acidic transcriptional activation domain of VP1 did not affect the inhibitory activity, indicating that VP1 has a discrete repressor function. Hence, physically combining activator and repressor functions in one protein may provide a mechanism to integrate the control of two normally consecutive developmental programs, seed maturation and seed germination.

The acidic transcriptional activation domains of herpes virus VP16 and yeast HAP4 have different co-factor requirements

The acidic transcriptional activation domain of the herpes virus activator VP16 requires an accessory protein complex for function, termed an adaptor. Although the activation domain of the yeast activator HAP4 is also highly negatively charged, its function is independent of at least one component of the adaptor complex, ADA2. In this study, we have used an in vitro inhibition assay to determine whether the activation domains of VP16 and HAP4 use a similar mechanism to potentiate transcription. Both domains had potent activation ability, indicating a similar strength of action. However, the capacity of each domain to inhibit activation of a heterologous test promoter (dA/dT) was sharply dissimilar. VP16 selectively inhibited activated transcription of dA/dT, without affecting basal transcription, implying that VP16 and the activator protein of the dA/dT promoter share a mechanism for activation. In contrast, HAP4 was totally unable to inhibit activated transcription of the dA/dT template. In the second part of the study, a genetic selection was used to obtain mutations in putative cofactor genes for HAP4. The spectrum of phenotypes caused by these mutations was strikingly different than mutations in the adaptor for the VP16 activation domain. These results strongly suggest that HAP4 and VP16 have distinct cofactor requirements, although they are both acidic activators.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

The Equine Herpesvirus Type 1 Immediate-Early Gene Product Contains an Acidic Transcriptional Activation Domain

The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene product, an ICP4 homolog, is the major regulatory protein encoded by EHV-1 during cytolytic infection. The IE gene product has been demonstrated to induce reporter gene expression directed by both homologous and heterologous viral promoters, including the EHV-1 thymidine kinase (tk) promoter, the herpes simplex virus type 1 (HSV-1) tk and ICP4 promoters, and the simian virus 40 early promoter. In this report, the transcriptional activation domain of the EHV-1 IE gene product was mapped to within an acidic, 87-amino-acid region (amino acids 3 to 89) at the amino-terminus of the IE molecule. It is demonstrated that the IE transcriptional activation domain, when fused to the DNA-binding domain of the yeast transcriptional activator GAL4, can activate gene expression in cell lines derived from at least two different species. Moreover, it is shown that the EHV-1 IR2 gene product (Harty and O'Callaghan, J. Virol. 65, 3829-3838, 1991), a truncated form of the IE polypeptide lacking IE amino acid residues 1-322 (and, therefore lacks the deduced transcriptional activation domain), fails to transactivate the EHV-1 tk promoter, but retains the ability to down-regulate the EHV-1 IE promoter. Fusion of the acidic transcriptional activation domain of the HSV-1 virion protein VP16 to the transactivation-deficient IR2 gene product restored the ability of this truncated IE polypeptide to transactivate the EHV-1 tk promoter. These findings suggest a role for the IR2 protein as a trans -repressor of EHV-1 gene expression.

Herpes simplex virus VP16 forms a complex with the virion host shutoff protein vhs.

Herpes simplex virus (HSV) virions contain at least two regulatory proteins that modulate gene expression in infected cells: the transcriptional activator VP16 and the virion host shutoff protein vhs. VP16 stimulates transcription of the HSV immediate-early genes, and vhs suppresses host protein synthesis and induces accelerated turnover of cellular and viral mRNAs. We report here that vhs binds directly to VP16: vhs and VP16 were coprecipitated from infected cells by an anti-vhs antiserum, and vhs and VP16 protein A fusions each bound intact versions of the other protein in a solid-phase capture assay. In addition, vhs and VP16 interacted in the two-hybrid activator system when coexpressed in Saccharomyces cerevisiae. vhs residues 238 to 344 were sufficient for the interaction, and the VP16 acidic transcriptional activation domain was not required. vhs blocked the ability of VP16 to enter a multiprotein complex on an immediate-early TAATGARATTC consensus sequence, indicating that vhs interacts with one or more regions of VP16 required for promoter recognition. We suggest that this interaction may play a structural role in the assembly of HSV virions and modulate the activity of vhs during infection.