Gibberellin (GA(3))-responsive aleurone protoplasts isolated from Avena sativa have been successfully used as a transient expression system to analyse promoter fusions between the wheat alpha-amylase gene alpha-Amy2/54 and the reporter gene GUS. Following PEG-mediated uptake of plasmid DNA, transient expression directed by the alpha-Amy2/54 promoter was found to be regulated in the same way as the endogenous oat alpha-amylase genes. Expression was thus dependent on the inclusion of GA(3) in the protoplast incubation media, could not be detected before a lag phase of 2 days following transformation and was inhibited by simultaneous addition of abscisic acid (ABA) with GA(3) to the media. In contrast, expression from the CaMV 35S promoter in the same system was not affected by GA(3) or ABA and could be detected 1 day after transformation. Introduction of a further three different promoters into the aleurone protoplasts confirmed that GA(3) specifically controlled transient expression from the alpha-Amy2/54 promoter only. Promoter deletions of the alpha-Amy2/54: GUS fusion demonstrated that sequences within 300 bp of the start of transcription of the gene were sufficient to direct high-level expression that was regulated by GA(3) and ABA.

The human immunodeficiency virus type I (HIV-1) transactivator protein Tat is an unusual transcriptional activator that is thought to act solely by promoting RNA polymerase II processivity. Here we study the mechanism of Tat action by analyzing transcription complex (TC) assembly in vivo using chromatin immunoprecipitation assays. We find, unexpectedly, that like typical activators Tat dramatically stimulates TC assembly. Surprisingly, however, the TC formed on the HIV-1 long terminal repeat is atypical and contains TATA-box-binding protein (TBP) but not TBP-associated factors (TAFs). Tat function involves direct interaction with the cellular cofactor positive transcription elongation factor b (P-TEFb). Artificial tethering of P-TEFb subunits to HIV-1 promoter DNA or nascent RNA indicates that P-TEFb is responsible for directing assembly of a TC containing TBP but not TAFs. On the basis of this finding, we identify P-TEFb-dependent cellular promoters that also recruit TBP in the absence of TAFs. Thus, in mammalian cells transcription of protein-coding genes involves alternative TCs that differ by the presence or absence of TAFs.

BACKGROUND: Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand. RESULTS: We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos. CONCLUSION: The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.

Myocardin is a transcriptional coactivator that regulates cardiac and smooth muscle gene expression by associating with serum response factor. We show that GATA transcription factors can either stimulate or suppress the transcriptional activity of myocardin, depending on the target gene. Modulation of myocardin activity by GATA4 is mediated by the physical interaction of myocardin with the DNA binding domain of GATA4 but does not require binding of GATA4 to DNA. Paradoxically, the transcription activation domain of GATA4 is dispensable for the stimulatory effect of GATA4 on myocardin activity but is required for repression of myocardin activity. The ability of GATA transcription factors to modulate myocardin activity provides a potential mechanism for fine tuning the expression of serum response factor target genes in a gene-specific manner.

Positive cofactor 4 (PC4) is a coactivator that strongly augments transcription by various activators, presumably by facilitating the assembly of the preinitiation complex (PIC). However, our previous observation of stimulation of promoter escape in GAL4-VP16-dependent transcription in the presence of PC4 suggested a possible role for PC4 in this step. Here, we performed quantitative analyses of the stimulatory effects of PC4 on initiation, promoter escape, and elongation in GAL4-VP16-dependent transcription and found that PC4 possesses the ability to stimulate promoter escape in response to GAL4-VP16 in addition to its previously demonstrated effect on PIC assembly. This stimulatory effect of PC4 on promoter escape required TFIIA and the TATA box binding protein-associated factor subunits of TFIID. Furthermore, PC4 displayed physical interactions with both TFIIH and GAL4-VP16 through its coactivator domain, and these interactions were regulated distinctly by PC4 phosphorylation. Finally, GAL4-VP16 and PC4 stimulated both initiation and promoter escape to similar extents on the promoters with three and five GAL4 sites; however, they stimulated promoter escape preferentially on the promoter with a single GAL4 site. These results provide insight into the mechanism by which PC4 permits multiply bound GAL4-VP16 to attain synergy to achieve robust transcriptional activation.

The expression of several muscle-specific genes is partially or completely regulated by MCAT elements, which bind members of the TEF family of transcription factors. TEF1 itself is unable to activate reporter plasmids bearing TEF1-binding sites, suggesting that additional bridging or co-activating factors are necessary to allow interaction of TEF1 with the transcriptional machinery. In addition, none of the known TEF genes are exclusively expressed in the cardiac or skeletal muscle lineage to account for the muscle-specific expression of MCAT-dependent genes. Here we describe that VITO-1, a new SID (scalloped interaction domain)-containing protein, binds to TEF1 in vitro and strongly stimulates transcription of a MCAT reporter plasmid together with TEF-1. Since VITO-1 is predominantly expressed in the skeletal muscle lineage, it might serve as an essential transcriptional intermediary factor to promote muscle-specific expression via MCAT cis-regulatory elements. Although VITO-1 alone is not sufficient to initiate myogenic conversion of 10T1/2 fibroblastic cells, it enhanced MyoD-mediated myogenic conversion. In addition, interference with VITO-1 expression by siRNA attenuated differentiation of C2C12 muscle cells and MyoD-dependent myogenesis in 10T1/2 cells. We conclude that VITO-1 is a crucial new cofactor of the muscle regulatory programme.

The ZAS proteins are large zinc-finger transcriptional proteins implicated in growth, signal transduction, and lymphoid development. Recombinant ZAS fusion proteins containing one of the two DNA-binding domains have been shown to bind specifically to the kappaB motif, but the endogenous ZAS proteins or their physiological functions are largely unknown. The kappaB motif, GGGACTTTCC, is a gene regulatory element found in promoters and enhancers of genes involved in immunity, inflammation, and growth. The Rel family of NF-kappaB, predominantly p65.p50 and p50.p50, are transcription factors well known for inducing gene expression by means of interaction with the kappaB motif during acute-phase responses. A functional link between ZAS and NF-kappaB, two distinct families of kappaB-binding proteins, stems from our previous in vitro studies that show that a representative member, ZAS3, associates with TRAF2, an adaptor molecule in tumor necrosis factor signaling, to inhibit NF-kappaB activation. Biochemical and genetic evidence presented herein shows that ZAS3 encodes major kappaB-binding proteins in B lymphocytes, and that NF-kappaB is constitutively activated in ZAS3-deficient B cells. The data suggest that ZAS3 plays crucial functions in maintaining cellular homeostasis, at least in part by inhibiting NF-kappaB by means of three mechanisms: inhibition of nuclear translocation of p65, competition for kappaB gene regulatory elements, and repression of target gene transcription.

Insulators are a class of elements that define independent domains of gene function. The Drosophila gypsy insulator is proposed to establish regulatory isolation by forming loop domains that constrain interactions between transcriptional control elements. This supposition is based upon the observation that insertion of a single gypsy insulator between an enhancer and promoter blocks enhancer function, while insertion of two gypsy insulators promotes enhancer bypass and activation of transcription. To investigate this model, we determined whether non-gypsy insulators interacted with each other and with the gypsy insulator. Pairs of scs or scs' insulators blocked enhancer function. Further, an intervening scs insulator did not block gypsy insulator interactions. Taken together, these data suggest that not all Drosophila insulators interact, with this property restricted to some insulators, such as gypsy. Three gypsy insulators inserted between an enhancer and promoter blocked enhancer function, indicating that gypsy insulator interactions may be restricted to pairs. Our studies imply that formation of loop domains may represent one of many mechanisms used by insulators to impart regulatory isolation.

Binding of transcriptional activators to specific sites on DNA, mediated by their DNA-binding domain, is a key regulatory point in transcriptional regulation. With a GFP-based microscopic assay, we investigated how the prototypical activator GAL4 effectively binds to a promoter in living yeast cells. We show that GAL4 relies on a previously unrevealed mechanism involving 'DNA-binding enhancers'(DBEs), the regions of GAL4 that assist DNA-binding domain association with DNA. GAL4 contains two DBEs, one, but not the other, physically overlapping the principal transcriptional activation domain. Either of the DBEs, however, can function independently of transcriptional activation, indicating a discrete mechanism responsible for DNA-binding enhancement. The effect of DBEs, while not limited to natural target promoters, is still not universal and can be profoundly affected by the binding-site context. The GAL4 DBEs can also enhance promoter binding of an unrelated DNA-binding domain, and possibly represent a new modular functional unit responsible for effective binding of diverse regulatory factors to DNA in vivo.

The developmental program of nodulation is regulated systemically in leguminous host species. A mutant astray (Ljsym77) in Lotus japonicus has lost some sort of its ability to regulate this symtem, and shows enhanced and early nodulation. In the absence of rhizobia, this mutant exhibits characteristics associated with defects in light and gravity responses. These nonsymbiotic phenotypes of astray are very similar to those observed in photomorphogenic Arabidopsis mutant hy5. Based on this evidence, we predicted that astray might contain a mutation in the HY5 homologue of L. japonicus. The homologue, named LjBzf, encodes a basic leucine zipper protein in the C-terminal half that shows the highest level of identity with HY5 of all Arabidopsis proteins. It also encodes legume-characteristic combination of motifs, including a RING-finger motif and an acidic region in the N-terminal half. The astray phenotypes were cosegregated with LjBzf, and the failure to splice the intron was detected. Nonsymbiotic and symbiotic phenotypes of astray were complemented by introduction of CaMV35SLjBzf. It is noteworthy that although Arabidopsis hy5 showed an enhancement of lateral root initiation, Lotus astray showed an enhancement of nodule initiation but not of lateral root initiation. Legume-characteristic combination of motifs of ASTRAY may play specific roles in the regulation of nodule development.

The repressor of bacteriophage lambda is a protein containing two domains of approximately equal size. Fragments containing the amino-terminal domain of repressor bind specifically to lambda operator DNA and mediate positive and negative control of lambda transcription both in vitro and in vivo.

Many crucial cellular enzymes--including RNA polymerases, kinases, phosphatases, proteases, acetylaters, etc.--have multiple potential substrates. Regulation entails substrate selection, a process effected by a mechanism we call regulated localization. This formulation is particularly well illustrated by the mechanisms of gene regulation. Analysis of these mechanisms reveals that regulated localization requires simple molecular interactions. These molecular interactions readily lend themselves to combinatorial control. This system of regulation is highly 'evolvable'. Its use accounts, at least in part, for the nature of many of the complexities observed in biological systems.

BACKGROUND: Most transcriptional activators minimally comprise two functional modules, one for DNA binding and the other for activation. Several activators also bear an oligomerization region and bind DNA as dimers or higher order oligomers. In a previous study we substituted these domains of a protein activator with synthetic counterparts [Mapp et al., Proc. Natl. Acad. Sci. USA 97 (2000) 3930-3935]. An artificial transcriptional activator, 4.2 kDa in size, comprised of a DNA binding hairpin polyamide tethered to a 20 residue activating peptide (AH) was shown to stimulate promoter specific transcription [Mapp et al., Proc. Natl. Acad. Sci. USA 97 (2000) 3930-3935]. The question arises as to the general nature and the versatility of this minimal activator motif and whether smaller ligands can be designed which maintain potent activation function. RESULTS: Here we have replaced the 20 amino acid AH peptide with eight or 16 residues derived from the activation domain of the potent viral activator VP16. The 16 residue activation module coupled to the polyamide activated transcription over two-fold better than the analogous AH conjugate. Altering the site of attachment of the activation module on the polyamide allowed reduction of the intervening linker from 36 atoms to eight without significant diminution of the activation potential. In this study we also exchanged the polyamide to target a different sequence without compromising the activation function further demonstrating the generality of this design. CONCLUSIONS: The polyamide activator conjugates described here represent a class of DNA binding ligands which are tethered to a second functional moiety, viz. an activation domain, that recruits elements of the endogenous transcriptional machinery. Our results define the minimal structural elements required to construct artificial, small molecule activators. If such activators are cell-permeable and can be targeted to designated sites in the genome, this series of conjugates may then serve as a tool to study mechanistic aspects of transcriptional regulation and eventually to modulate gene expression relevant to human diseases.

In yeast (Saccharomyces cerevisiae), transcriptional activators, such as Gal4 and Gal4-VP16, work ordinarily from sites located in the upstream activating sequence (UAS) positioned about 250 base pairs upstream of the transcription start site. In contrast to their behaviour in mammalian cells, however, such activators fail to work when positioned at distances greater than approximately 600-700 base pairs upstream, or anywhere downstream of the gene. Here we show that, in yeast, a gene bearing an enhancer positioned 1-2 kilobases downstream of the gene is activated if the reporter is linked to a telomere, but not if it is positioned at an internal chromosomal locus. These observations are explained by the finding that yeast telomeres form back-folding, or looped, structures. Because yeast telomeric regions resemble the heterochromatin found in higher eukaryotes, these findings might also explain why transcription of some higher eukaryotic genes depends on their location in heterochromatin.

The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C(2) symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression.

Unlike the DNA-binding domains (DBD) of most eukaryotic transcription factors, Escherichia coli LacI family transcription factors are unable to bind to specific target DNA sequences without a cofactor-binding domain. In the present study, we reconstructed a novel DBD designated as PurHG, which binds constitutively to a 16bp purine repressor operator, by fusion of the purine repressor (PurR) DBD (residues 1-57) and the GAL4 dimerization domain (DD, residues 42-148). Binding of PurHG to DNA requires the dimerization and a hinge helix of PurR DBD. When the PurHG was expressed as a fusion protein in a form of a transcription activator (PurAD) or an artificial nuclear receptor (PurAPR or PurAER) responding to ligand, such as RU486 or beta-estradiol, it could regulate the expression of the reporter genes in NIH3T3 cells. The prerequisite region of the GAL4 DD for DNA-binding was amino acid residues from 42 to 98 in the form of PurAD, while the amino acid residues from 42 to 75 were sufficient for ligand-dependent regulation in the form of PurAPR. These results suggest that the dimerization function of the progesterone ligand-binding domain could be substituted for region 76-98 of the GAL4 DD. In summary, the fusion of the PurR DBD and the GAL4 DD generates fully active DNA-binding protein, PurHG, in vitro and in vivo, and these results provide the direct evidence of structural predictions that the proximate positioning of PurR hinge helical regions is critical for DNA-binding.

BACKGROUND: Mutations and gene expression alterations in brain tumors have been extensively investigated, however the causes of brain tumorigenesis are largely unknown. Animal models are necessary to correlate altered transcriptional activity and tumor phenotype and to better understand how these alterations cause malignant growth. In order to gain insights into the in vivo transcriptional activity associated with a brain tumor, we carried out genome-wide microarray expression analyses of an adult brain tumor in Drosophila caused by homozygous mutation in the tumor suppressor gene brain tumor (brat). RESULTS: Two independent genome-wide gene expression studies using two different oligonucleotide microarray platforms were used to compare the transcriptome of adult wildtype flies with mutants displaying the adult bratk06028 mutant brain tumor. Cross-validation and stringent statistical criteria identified a core transcriptional signature of brat(k06028) neoplastic tissue. We find significant expression level changes for 321 annotated genes associated with the adult neoplastic brat(k06028) tissue indicating elevated and aberrant metabolic and cell cycle activity, upregulation of the basal transcriptional machinery, as well as elevated and aberrant activity of ribosome synthesis and translation control. One fifth of these genes show homology to known mammalian genes involved in cancer formation. CONCLUSION: Our results identify for the first time the genome-wide transcriptional alterations associated with an adult brain tumor in Drosophila and reveal insights into the possible mechanisms of tumor formation caused by homozygous mutation of the translational repressor brat.

Like other nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) use a wide variety of protein-protein interactions to properly regulate transcription of target genes. In an attempt to identify novel PPAR-interacting proteins, a cDNA expression library was screened with bacterially expressed PPARalpha. One of the genes identified as a PPARalpha-associated protein by interaction cloning was the CREB-binding protein/p300-interacting transactivator with ED-rich tail 2 (CITED2, also called p35srj/mrg1/msg1). This coactivator interacted directly with PPARalpha in the presence or absence of ligand predominantly via the ligand binding domain of the nuclear receptor. In transient transfection reporter assays, CITED2 acted as a dose-dependent coactivator of PPARalpha-dependent transcriptional regulation in the presence of several exogenous ligands. CITED2 also increased PPARgamma-dependent regulation of reporter genes but had no effect on PPARbeta activity. To determine whether CITED2 affects endogenous gene expression, this protein was stably overexpressed (CITED2+) or repressed by small inhibitor RNA (CITED2-) in immortalized mouse hepatocytes. Relative to the control stably transfected or CITED2-cells, CITED2+ cells had an increased rate of cell proliferation. Microarray analysis and real time PCR showed that several genes are differentially affected by PPARalpha ligands in CITED2+ versus CITED2-cells. Genes that were affected by PPARalpha ligands in a CITED2-modulatory manner include angiopoietin-like protein 4, forkhead C2, hypoxia-inducible factor-1alpha, and MAPK phosphatase 1. Interestingly these genes share common functions in that they are known to promote vascularization and angiogenesis in response to hypoxia. The results described here suggest that CIT-ED2 is a coactivator of PPARalpha and that both proteins may participate in signaling cascades of hypoxic response and angiogenesis.

In eukaryotes, transcription of the diverse array of tens of thousands of protein-coding genes is carried out by RNA polymerase II. The control of this process is predominantly mediated by a network of thousands of sequence-specific DNA binding transcription factors that interpret the genetic regulatory information, such as in transcriptional enhancers and promoters, and transmit the appropriate response to the RNA polymerase II transcriptional machinery. This review will describe some early advances in the discovery and characterization of the sequence-specific DNA binding transcription factors as well as some of the properties of these regulatory proteins.

We previously demonstrated that a 31-bp nucleotide sequence located upstream of the rice Wx gene played an important role in its expression. We further showed that this cis-acting regulator interacts with nuclear proteins extracted from developing rice endosperm. We used the 31-bp sequence as bait in a yeast one-hybrid system to isolate several cDNA clones from a rice cDNA expression library. One of these cDNAs encodes a MYC protein, designated OsBP-5, which is 335 amino acids long and contains a putative basic helix-loop-helix-ZIP DNA-binding domain. This domain exhibits 50% amino acid sequence identity with the R/B proteins that regulate the expression of genes involved in anthocyanin biosynthesis in plants. The results of electrophoretic mobility shift assays (EMSAs) and Southwestern gel blots indicate that this protein binds specifically to the CAACGTG motif within the 31-bp sequence. However, by itself, the OsBP-5 protein is unable to trans-activate a lacZ reporter gene controlled by the 31-bp sequence when tested in a yeast expression system. Interestingly, OsBP-5 can trans-activate this reporter gene when another protein, OsEBP-89, a member of the EREBP family of transcription factors, is present. Furthermore, in vitro pull-down experiments show that a protein isolated from developing rice endosperm interacts with the OsBP-5 protein, and Western blots confirm that the interacting protein is OsEBP-89. The formation of a supershift band in EMSAs also indicates that two proteins interact with each other. Interference of OsBP-5 gene expression by double-stranded RNA reduces the amylose content in mature seed of transgenic rice plants but has no visible effect on their phenotype. These results suggest that the OsBP-5 and OsEBP-89 proteins act synergistically, perhaps as a heterodimer, to regulate the transcription of the rice Wx gene.

For many, if not most genes, the initiation of transcription is the principle point at which their expression is regulated. Transcription factors, some of which bind to specific DNA sequences, generally either activate or repress promoter activity and thereby control transcription initiation. Recent work has revealed in molecular detail some of the mechanisms used by transcription factors to bring about transcriptional repression. Some transcriptional repressor proteins counteract the activity of positively acting transcription factors. Other repressors inhibit the basal transcription machinery. In addition, the repression of transcription is often intimately associated with chromatin re-organisation. Many transcriptional repressor proteins interact either directly or indirectly with proteins that remodel chromatin or can themselves influence chromatin structure. This review discusses the mechanisms by which transcriptional repression is achieved and the role that chromatin re-organisation plays in this process.

DNA-binding transcription factors regulate the expression of genes near to where they bind. These factors can be activators or repressors of transcription, or both. Thus, a fundamental question is what determines whether a transcription factor acts as an activator or a repressor? Previous research into this question found that a protein's regulatory function is determined by one or more of the following factors: protein-protein contacts, position of the DNA-binding domain in the protein primary sequence, altered DNA structure, and the position of its binding site on the DNA relative to the transcription start site. Although there are many aspects specific to different transcription factors, in this work we demonstrate that, in general, in the prokaryote Escherichia coli, a transcription factor's protein family is not indicative of its regulatory function, but the position of its binding site on the DNA is.

The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication. Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication. The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes. In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73alpha up-regulates the activity of this promoter. Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73alpha-induced p21 promoter activity. Using deletion mutants, we found that the DNA-binding domains of both p53 and p73alpha are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively. CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73alpha. These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance.