Constitutive Activation Domain Research Articles

Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants.

The Agrobacterium tumefaciens VirG response regulator of the VirA/VirG two-component system was adapted to function in tobacco protoplasts. The subcellular localization of VirG and VirA proteins transiently expressed in onion cells was determined using GFP fusions. Preliminary studies using Gal4DBD-VP16 fusions with VirG and Escherichia coli UhpA, and NarL response regulators indicated compatibility of these bacterial proteins with the eukaryotic transcriptional apparatus. A strong transcriptional activator based on tandem activation domains from the Drosophila fushi tarazu and Herpes simplex VP16 was created. Selected configurations of the two-site Gal4-vir box GUS reporters were activated by chimeric effectors dependent on either the yeast Gal4 DNA-binding domain or that of VirG. Transcriptional induction of the GUS reporter was highest for the VirE19-element promoter with both constitutive and wild-type VirG-tandem activation domain effectors. Multiple VirE19 elements increased the reporter activity proportionately, indicating that the VirG DNA binding domain was functional in plants. The VirG constitutive-Q-VP16 effector was more active than the VirG wild-type. In both the constitutive and wild-type forms of VirG, Q-VP16 activated transcription of the GUS reporter best when located at the C-terminus, i.e. juxtaposed to the VirG DNA binding domain. These results demonstrate the possibility of using DNA binding domains from bacterial response regulators and their cognate binding elements in the engineering of plant gene expression.

3C Protease of Enterovirus D68 Inhibits Cellular Defense Mediated by Interferon Regulatory Factor 7.

Human enterovirus 68 (EV-D68) is a member of the EV-D species, which belongs to the EV genus of the Picornaviridae family. Over the past several years, clusters of EV-D68 infections have occurred worldwide. A recent outbreak in the United States is the largest one associated with severe respiratory illness and neurological complication. Although clinical symptoms are recognized, the virus remains poorly understood. Here we report that EV-D68 inhibits innate antiviral immunity by downregulation of interferon regulatory factor 7 (IRF7), an immune factor with a pivotal role in viral pathogenesis. This process depends on 3C(pro), an EV-D68-encoded protease, to mediate IRF7 cleavage. When expressed in host cells, 3C(pro) targets Q167 and Q189 within the constitutive activation domain, resulting in cleavage of IRF7. Accordingly, wild-type IRF7 is fully active. However, IRF7 cleavage abrogated its capacity to activate type I interferon expression and limit replication of EV-D68. Notably, IRF7 cleavage strictly requires the protease activity of 3C(pro). Together, these results suggest that a dynamic interplay between 3C(pro) and IRF7 may determine the outcome of EV-D68 infection. EV-D68 is a globally emerging pathogen, but the molecular basis of EV-D68 pathogenesis is unclear. Here we report that EV-D68 inhibits innate immune responses by targeting an immune factor, IRF7. This involves the 3C protease encoded by EV-D68, which mediates the cleavage of IRF7. These observations suggest that the 3C(pro)-IRF7 interaction may represent an interface that dictates EV-D68 infection.

Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses

Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses.

Dissection of Drosophila MTF-1 reveals a domain for differential target gene activation upon copper overload vs. copper starvation

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein conserved from mammals to insects. It mediates protection against heavy metal load by activating the expression of metallothionein and other genes. In Drosophila, MTF-1 serves a dual function in that it not only helps to protect against heavy metal load but also induces the expression of Ctr1B, the gene for an intestinal copper importer, upon copper starvation. By dissecting Drosophila MTF-1 function, we have identified determinants for nuclear import and export, and characterized a phosphorylation site mutant (T127A) that differentially affects MTF-1 target genes. Further, by generating a series of fusion proteins with the heterologous DNA binding domain of Gal4 we identified a strong, constitutive activation domain in the central region of MTF-1 (aa 352–540). By contrast, an extended fusion protein that includes MTF-1's C-terminus (aa 352–791) is not active in standard conditions but induced by copper load. The paramount regulatory importance of the C-terminal part, that harbors a cysteine-rich “metallothionein-like” domain, was corroborated by different experiments. Transgenic flies expressing C-terminally truncated MTF-1 variants displayed high constitutive transcription of both, the genes for metallothioneins and the copper importer Ctr1B. The indiscriminate activation of these genes that are normally induced under opposite conditions of copper load and copper starvation manifested itself in a shortened lifespan, crippled wings, and female sterility.

GeBP/GPL transcription factors regulate a subset of CPR5-dependent processes.

The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.

Activation of CREB/ATF Sites by Polyomavirus Large T Antigen

Polyomavirus large T antigen (LT) has a direct role in viral replication and a profound effect on cell phenotype. It promotes cell cycle progression, immortalizes primary cells, blocks differentiation, and causes apoptosis. While much of large T function is related to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family, we have previously shown that activation of the cyclin A promoter can occur through a non-Rb-dependent mechanism. Here we show that activation occurs via an ATF/CREB site. Investigation of the mechanism indicates that large T can synergize with CREB family members to activate transcription. Experiments with Gal4-CREB constructs show that synergy is independent of CREB phosphorylation by protein kinase A. Examination of synergy with Gal4-CREB deletion constructs indicates that large T acts on the constitutive activation domain of CREB. Large T can bind to CREB in vivo. Genetic analysis shows that the DNA-binding domain (residues 264 to 420) is sufficient to activate transcription when it is localized to the nucleus. Further analysis of the DNA-binding domain shows that while site-specific DNA binding is not required, non-site-specific DNA binding is important for the activation. Thus, CREB binding and DNA binding are both important for large T activation of CREB/ATF sites. In contrast to previous models where large T transactivation occurred indirectly, these results also suggest that large T can act directly at promoters to activate transcription.

Analysis of the spacing between the two palindromes of activation sequence-1 with respect to binding to different TGA factors and transcriptional activation potential.

In higher plants, activation sequence-1 (as-1) of the cauliflower mosaic virus 35S promoter mediates both salicylic acid- and auxin-inducible transcriptional activation. Originally found in viral and T-DNA promoters, as-1-like elements are also functional elements of plant promoters activated in the course of a defence response upon pathogen attack. as-1-like elements are characterised by two imperfect palindromes with the palindromic centres being spaced by 12 bp. They are recognised by plant nuclear as-1-binding factor ASF-1, the major component of which is basic/leucine zipper (bZIP) protein TGA2.2 (approximately 80%) in Nicotiana tabacum. In electrophoretic mobility shift assays, ASF-1 as well as bZIP proteins TGA2.2, TGA2.1 and TGA1a showed a 3-10-fold reduced binding affinity to mutant as-1 elements encoding insertions of 2, 4, 6, 8 or 10 bp between the palindromes, respectively. This correlated with a 5-10-fold reduction in transcriptional activation from these elements in transient expression assays. Although ASF-1 and TGA factors bound efficiently to a mutant element carrying a 2 bp deletion between the palindromes [as-1/(-2)], the latter was strongly compromised with respect to mediating gene expression in vivo. A fusion protein consisting of TGA2.2 and a constitutive activation domain mediated transactivation from as-1/(-2) demonstrating binding of TGA factors in vivo. We therefore conclude that both DNA binding and transactivation require optimal positioning of TGA factors on the as-1 element.

Mechanisms of basal and kinase-inducible transcription activation by CREB

The cAMP response element (CRE)-binding protein (CREB) stimulates basal transcription of CRE-containing genes and mediates induction of transcription upon phosphorylation by protein kinases. The basal activity of CREB maps to a carboxy-terminal constitutive activation domain (CAD), whereas phosphorylation and inducibility map to a central, kinase-inducible domain (KID). The CAD interacts with and recruits the promoter recognition factor TFIID through an interaction with a specific TATA-binding-protein-associated factor (TAF), dTAFII110/ hTAFII135. Interaction between the TAF and the CAD is mediated by a central cluster of hydrophobic amino acids, mutation of which disrupts TAF binding, polymerase recruitment, and transcription activation. Assessment of the contributions of the CAD and KID to recruitment of the polymerase complex versus enhancement of subsequent reaction steps (isomerization, promoter clearance, and reinitiation) showed that the CAD and P-KID act in a concerted mechanism to stimulate transcription. The CAD, but not the KID, mediated recruitment of a complex containing components of a transcription initiation complex, including pol II, IIB, and IID. However, the CAD was relatively ineffective in stimulating subsequent steps in the reaction mechanism. In contrast, phosphorylation of the KID in CREB effectively stimulated isomerization of the recruited polymerase complex and multiple-round transcription. A model for the activation of transcription by phosphorylated CREB is proposed, in which the polymerase is recruited by interaction of the CAD with TFIID and the recruited polymerase is activated further by phosphorylation of the KID in CREB.

Overexpression of Pti5 in tomato potentiates pathogen-induced defense gene expression and enhances disease resistance to Pseudomonas syringae pv. tomato.

The tomato Pti5 gene encodes a pathogen-inducible ethylene response element-binding protein-like transcription factor that interacts with the disease resistance gene product Pto. Overexpression of Pti5 or Pti5-VP16, a translational fusion with a constitutive transcriptional activation domain, in tomato enhanced resistance to Pseudomonas syringae pv. tomato. Constitutive expression of Pti5 or Pti5-VP16 did not affect the basal level of pathogenesis-related gene expression, but it accelerated pathogen-induced expression of GluB and Catalase. The results demonstrate a positive role of Pti5 in defense gene regulation and disease resistance and suggest that a pathogen-activated posttranscriptional regulatory step is necessary for the pathogen induction of the defense gene expression.

The coactivator dTAF(II)110/hTAF(II)135 is sufficient to recruit a polymerase complex and activate basal transcription mediated by CREB.

A specific TATA binding protein-associated factor (TAF), dTAF(II)110/hTAF(II)135, interacts with cAMP response element binding protein (CREB) through its constitutive activation domain (CAD), which recruits a polymerase complex and activates transcription. The simplest explanation is that the TAF is a coactivator, but several studies have questioned this role of TAFs. Using a reverse two-hybrid analysis in yeast, we previously mapped the interaction between dTAF(II)110 (amino acid 1-308) and CREB to conserved hydrophobic amino acid residues in the CAD. That mapping was possible only because CREB fails to activate transcription in yeast, where all TAFs are conserved, except for the TAF recognizing CREB. To test whether CREB fails to activate transcription in yeast because it lacks a coactivator, we fused dTAF(II)110 (amino acid 1-308) to the TATA binding protein domain of the yeast scaffolding TAF, yTAF(II)130. Transformation of yeast with this hybrid TAF conferred activation by the CAD, indicating that interaction with yTFIID is sufficient to recruit a polymerase complex and activate transcription. The hybrid TAF did not mediate activation by VP16 or vitamin D receptor, each of which interacts with TFIIB, but not with dTAF(II)110 (amino acid 1-308). Enhancement of transcription activation by dTAF(II)110 in mammalian cells required interaction with both the CAD and TFIID and was inhibited by mutation of core hydrophobic residues in the CAD. These data demonstrate that dTAF(II)110/hTAF(II)135 acts as a coactivator to recruit TFIID and polymerase and that this mechanism of activation is conserved in eukaryotes.

Recruitment of an RNA polymerase II complex is mediated by the constitutive activation domain in CREB, independently of CREB phosphorylation.

The cAMP response element binding protein (CREB) is a bifunctional transcription activator, exerting its effects through a constitutive activation domain (CAD) and a distinct kinase inducible domain (KID), which requires phosphorylation of Ser-133 for activity. Both CAD and phospho-KID have been proposed to recruit polymerase complexes, but this has not been directly tested. Here, we show that the entire CREB activation domain or the CAD enhanced recruitment of a complex containing TFIID, TFIIB, and RNA polymerase II to a linked promoter. The nuclear extracts used mediated protein kinase A (PKA)-inducible transcription, but phosphorylation of CRG (both of the CREB activation domains fused to the Gal4 DNA binding domain) or KID-G4 did not mediate recruitment of a complex, and mutation of the PKA site in CRG abolished transcription induction by PKA but had no effect upon recruitment. The CREB-binding protein (CBP) was not detected in the recruited complex. Our results support a model for transcription activation in which the interaction between the CREB CAD and hTAFII130 of TFIID promotes the recruitment of a polymerase complex to the promoter.

Multiple Regulatory Domains Control IRF-7 Activity in Response to Virus Infection

Recent studies implicate the interferon regulatory factors (IRF), IRF-3 and IRF-7, as key activators of Type 1 interferon genes, as well as the RANTES (regulated on activation normal T cell expressed) chemokine gene. Both IRF-3 and IRF-7 are regulated in part by virus-induced C-terminal phosphorylation, leading to nuclear translocation, stimulation of DNA binding, and transcriptional activities. Structure-function studies with IRF-7 suggested a complex organization of the C-terminal region, with a constitutive activation domain located between amino acids 150-246, an accessory inducibility region at the very end of IRF-7 between amino acids 467 and 503, and an inhibitory region (amino acids 341-467) adjacent to the C-terminal end that interferes with transactivation. Furthermore, an element that increases basal and virus-inducible activity is located between amino acids 278 and 305. A transcriptionally active form of IRF-7 was also generated by substitution of Ser-477 and Ser-479 residues with the phosphomimetic Asp. IRF-7, particularly IRF-7(S477D/S479D), was a strong transactivator of type I interferon and RANTES chemokine gene expression. Unlike wild type IRF-3, IRF-7 overexpression was able to stimulate inteferon gene expression in the absence of virus infection. Using tagged versions of IRF-7 and IRF-3, the formation of homo- and heterodimers was detected by co-immunoprecipitation. These results demonstrate that IRF-3 and IRF-7 transcription factors possess distinct structural characteristics that impart complementary rather than redundant functional roles in cytokine gene activation.

Distinct cAMP response element-binding protein (CREB) domains stimulate different steps in a concerted mechanism of transcription activation.

Hormones and neurotransmitters rapidly change patterns of gene expression in target cells by activating protein kinases that phosphorylate and modify the activity of CREB and other transcription factors. Although CREB was initially characterized as mediating the response to cAMP, CREB phosphorylation and activation are stimulated by diverse extracellular signals and protein kinases in essentially all cells and tissues. CREB stimulates transcription through a constitutive activation domain (CAD), which interacts with the promoter recognition factor TFIID, and through a kinase-inducible domain (KID), when Ser-133 is phosphorylated. The present study provides new insight into the mechanism of activation by showing that each of the CREB domains contributes to transcription initiation by stimulating sequential steps in the transcription reaction. The CAD effectively assembled a polymerase complex, as evidenced by constitutive activation in vivo and stimulation of single-round transcription in vitro. In contrast, phosphorylation of the KID in CREB stimulated isomerization of the polymerase complex, as determined by abortive initiation, and promoter clearance and/or reinitiation, as measured by multiple rounds of transcription. Our results provide evidence for a new model for CREB-mediated induction through a concerted mechanism involving establishment of a polymerase complex by the CAD, followed by stimulation of isomerization, promoter clearance, and/or reinitiation by phosphorylated KID to enhance target gene transcription.

Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, growth with a non-fermentable carbon source requires co-ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source-responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO2TAD) and/or a carbon source-independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE-dependent synthetic minimal promoter showed a 40-fold derepression with wild-type CAT8, an almost constitutive expression was found with a MET25-CAT8-INO2TAD fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source-dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.

Coactivators for the orphan nuclear receptor RORalpha.

A mutation in the nuclear orphan receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have shown previously that RORalpha contains a strong constitutive activation domain in its C terminus. We therefore searched for mammalian RORalpha coactivators using the minimal activation domain as bait in a two-hybrid screen. Several known and putative coactivators were isolated, including glucocorticoid receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205). These interactions were confirmed in vitro and require the intact activation domain of RORalpha although different requirements for interaction with GRIP-1 and PBP were detected. Even in the absence of exogenous ligand, RORalpha interacts with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be present in these complexes. Thus we have identified several potential RORalpha coactivators that, in contrast to the interactions with hormone receptors, interact with RORalpha in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a coactivator for the RORalpha both in yeast and in mammalian cells. Thus, GRIP-1 is the first proven coactivator for RORalpha.

The CREB Constitutive Activation Domain Interacts with TATA-binding Protein-associated Factor 110 (TAF110) through Specific Hydrophobic Residues in One of the Three Subdomains Required for Both Activation and TAF110 Binding

The cAMP response element-binding protein (CREB) mediates both basal and PKA-inducible transcription through two separate and independently active domains, the constitutive activation domain (CAD) and the kinase-inducible domain, respectively. The CREB CAD interacts with the general transcription factor TFIID through one or more of the TATA-binding protein-associated factors (TAFs), one of which is TAF110. The CAD is composed of three subdomains, rich in either serine, hydrophobic amino acids, or glutamine. In the present study, analysis of deletion mutants of the CAD showed that all three CAD subdomains were required for effective interaction with TAF110 in a yeast two-hybrid assay. Therefore, a library of random point mutations within the CAD was analyzed in a reverse two-hybrid screen to identify amino acids that are essential for interaction with the TAF. Interaction defects resulted solely from mutations of hydrophobic amino acid residues within the hydrophobic cluster to charged amino acid residues. Together, the deletion and mutation analyses suggest that the entire CAD provides an environment for a specific hydrophobic interaction with TAF110 that is crucial for interaction. Our results provide further evidence for a model of basal activation by CREB involving interaction with TAF110 that promotes recruitment or stabilization of TFIID binding to the promoter, which facilitates pre-initiation complex assembly.

Activity regulation of a Hox protein and a role for the homeodomain in inhibiting transcriptional activation.

Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). We present results supporting a novel mechanism for Hox cofactor function: regulation of transcriptional activation function. First, we provide evidence that the Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. Secondly, either Dfd or a Dfd-VP16 hybrid mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independently of Exd enhancement of Dfd-binding affinity on the composite site. Thirdly, transfection assays confirmed that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family.

Activation of Hypoxia-inducible Factor-1; Definition of Regulatory Domains within the α Subunit

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

Hormone-dependent Transactivation by Estrogen Receptor Chimeras That Do Not Interact with hsp90: EVIDENCE FOR TRANSCRIPTIONAL REPRESSORS

The ligand-free estrogen receptor (ER), like other steroid receptors, interacts with the 90-kDa heat shock protein hsp90 in vitro. Analysis of the effect of potential ER-hsp90 interactions in vivo on receptor function is complicated by the fact that hsp90 binds to ER domains required for hormone binding and stable DNA binding. ER chimeras were therefore created by replacing the ER DNA binding domain with that of GAL4. In addition, the N-terminal AF-1 domain of the ER was replaced with the strong constitutive activation domain of VP16 to create VP16-GAL-ERs. These chimeras bind DNA in a ligand-independent manner, but, importantly, are ligand-dependent transactivators, unlike VP16-GAL, which displays strong constitutive activity under the same conditions. Hormone induces transactivation by VP16-GAL-ERs to levels similar to the constitutive activity of VP16-GAL. Glycerol gradient and coimmunoprecipitation experiments showed that, unlike the wild-type ER, VP16-GAL-ER chimeras do not interact with hsp90. Deletion analyses indicate that a region of the ER, primarily between amino acids 370 and 470, is responsible for repressed transcription. Our results suggest that interaction with hsp90 is not necessary for controlling hormone-dependent transcription by the ER and provide evidence for repressor factors that interact with the N-terminal portion of the receptor's ligand binding domain in the absence of hormone.

CAMP Response Element-binding Protein (CREB) Interacts with Transcription Factors IIB and IID

cAMP response element-binding protein (CREB) participates in both constitutive and cAMP-induced transcription of cAMP-responsive genes. CREB-mediated constitutive transcription requires only CREB-binding sites and a minimal promoter region (containing the TATA through start sequences), indicating that CREB interacts directly with components of the general transcription machinery. In this study, a coimmunoprecipitation assay was used to test for interaction of CREB with the general transcription factors (TF) TFIIB and TFIID and the core component of TFIID, TATA-binding protein (TBP). Human TFIIB and TBP, tagged with distinct epitopes (eTFIIB and eTBP), were expressed in and purified from Escherichia coli, and holo-eTFIID, containing eTBP, was obtained from the HeLa cell line LTR alpha 3. 35S-Labeled CREB, synthesized in vitro and incubated with eTFIIB, was coimmunoprecipitated with antibody recognizing eTFIIB, indicating that CREB specifically binds to TFIIB. 35S-CREB was coimmunoprecipitated with antibody against eTBP, but only when incubated with the holo-eTFIID complex, not with eTBP alone. TFIIB interacted with TBP, but CREB was not coprecipitated with the eTBP antibody when incubated with eTBP plus TFIIB, so CREB did not form a stable ternary complex with TFIIB and TBP. Conversely, depletion of TFIIB from the holo-TFIID preparation did not diminish the level of interaction between CREB and TFIID. Thus, CREB interacts independently with TFIIB and TFIID, but not directly with TBP. A protein kinase A phosphorylation site mutant of CREB and wild-type CREB exhibited equivalent interaction with TFIIB, indicating that this phosphorylation is not required. Consistent with the role of CREB in promoting constitutive or basal transcription, the constitutive activation domain of CREB was sufficient for interaction with both TFIIB and TFIID.