Glutamine-rich Domain Research Articles

Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes.

Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins.

The PRDI-BF1/Blimp-1 protein is a transcriptional repressor required for normal B-cell differentiation, and it has been implicated in the repression of beta-interferon (IFN-beta) and c-myc gene expression. Here, we show that PRDI-BF1 represses transcription of the IFN-beta promoter and of an artificial promoter through an active repression mechanism. We also identified a minimal repression domain in PRDI-BF1 that is sufficient for transcriptional repression when tethered to DNA as a Gal4 fusion protein. Remarkably, this repression domain interacts specifically with hGrg, TLE1, and TLE2 proteins, all of which are members of the Groucho family of transcriptional corepressors. In addition, the hGrg protein itself can function as a potent repressor when tethered to DNA through the Gal4 DNA-binding domain. We also find that the amino-terminal glutamine-rich domains of hGrg and TLE1 are sufficient to mediate dimerization of the two Groucho family proteins. Proteins containing only this domain can function as a dominant-negative inhibitor of PRDI-BF1 repression, and can significantly increase the IFN-beta promoter activity after virus induction. We conclude that PRDI-BF1/Blimp-1 represses transcription by recruiting a complex of Groucho family proteins to DNA, and suggest that such corepressor complexes are required for the postinduction repression of the IFN-beta promoter.

DNA distortion and multimerization: novel functions of the glutamine-rich domain of GAGA factor

GAGA factor (GAF) is an essential protein in Drosophila melanogaster, important for the transcriptional regulation of numerous genes. The effect of GAF on chromatin structure and promoter function has been the subject of much attention, yet little is known of the actual mechanism and the specific contributions of individual GAF domains to its function. The DNA-binding activity of GAF, as specified by the single zinc finger binding domain (Zn), has been examined in some detail; however, the functions of the POZ/BTB and glutamine domain (Q) remain poorly understood. Here, we report three separate activities of the Q domain of GAF; promoter distortion, single-strand binding, and multimerization. In vitro, GAF binding to the hsp70 promoter produces extended DNase I protection and KMnO4 hypersensitivity. These activities require both the Zn domain and Q domain of GAF, and appear independent of the POZ/BTB domain. GAF also has a single-stranded DNA binding affinity, as does the Q-rich region alone. GAF forms multimers both in vitro and in vivo, and the Q domain itself forms multimers. Protein-protein interactions mediated by the Q domain may, therefore, be at least partially responsible for the multimerization capabilities of GAF. We discuss these findings in the context of their possible function in GAF mediated transcriptional regulation.

The glutamine-rich domain of the Drosophila GAGA factor is necessary for amyloid fibre formation in vitro, but not for chromatin remodelling

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in β-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.

Nerve Growth Factor Up-regulates the Transcriptional Activity of CBP through Activation of the p42/p44MAPK Cascade

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.

Cloning and Mapping of ZNF231, a Novel Brain-Specific Gene Encoding Neuronal Double Zinc Finger Protein Whose Expression Is Enhanced in a Neurodegenerative Disorder, Multiple System Atrophy (MSA)

A novel brain-specific gene, neuronal double zinc finger protein (ZNF231), was cloned and mapped. We used the high-density cDNA filter method to analyze the gene-expression profile in brains with multiple system atrophy (MSA). MSA is a sporadic progressive neurodegenerative disease characterized clinically by cerebellar symptoms, parkinsonism, autonomic dysfunction, or their various combinations, but its pathogenesis has yet to be clarified. In total, 8300 cDNA clones were screened, and a novel gene, ZNF231, was identified, whose expression was elevated in cerebella of patients with MSA. Its transcript is approximately 16 kb long and encodes an open reading frame of 3926 amino acid residues that has several interesting motifs; two glycine–proline dipeptide repeats (aa 22–32 and aa 61–74), a pair of homologous C8 double zinc finger motifs (aa 169–226 and aa 465–521), a leucine zipper motif (aa 561–582), a SH3 domain-binding motif (aa 825–831), two nuclear targeting signals (aa 1011–1028 and aa 1071–1091), two glutamine-rich domains (aa 2428–2473 and aa 3775–3804), and a histidine-rich domain (aa 3597–3682). These features suggest that the new gene encodes a nuclear protein or transcription regulator. Northern blot and RT-PCR analyses showed that its expression is specific to the brain and apparently restricted to the neurons. Elevation of ZNF231 expression may be involved in the pathogenesis of multiple system atrophy. The gene for ZNF231 is located on chromosome 3p21.

Glutamine-rich Domains Activate Transcription in YeastSaccharomyces cerevisiae

Activation domains of eukaryotic transcription factors can be classified into at least three distinct types based on their amino acid composition: acidic, proline-rich, and glutamine-rich. Acidic activators, such as yeast GAL4 and GCN4 and herpes simplex virus VP16, have been shown to stimulate transcription in various higher and lower eukaryotic cells. Similarly, proline-rich activators also function in both mammalian and yeast cells. These activators are regarded to possess "universal" activating potentials. By contrast, several studies have suggested that glutamine-rich activators such as human Sp1 are active in higher (mammalian) but not lower (yeast) eukaryotic cells. One interpretation is that lower eukaryotic cells lack a critical co-factor necessary for a glutamine-rich domain. This reasoning is counter-intuitive because many native yeast activator proteins contain glutamine-rich domains. Here, we have investigated the activity of a glutamine-rich GAL4-Sp1 domain A (Sp1A) hybrid protein in yeast Saccharomyces cerevisiae. We show that GAL4-Sp1A activated a GAL1-lacZ reporter by more than 200-fold over basal when the reporter was carried on a 2mu vector. The generality of the Sp1A results is supported by our finding that yeast glutamine-rich domains from HAP2 and MCM1 are also transcriptionally active in S. cerevisiae. Interestingly, we found that glutamine-rich domains are considerably less potent when responsive promoters (i.e. GAL1-lacZ) are integrated into yeast chromosome. Thus our results segregate the inherent transcriptional activity of a glutamine-rich domain in yeast S. cerevisiae from its apparent lack of activity when assayed on chromosomally embedded promoters.

TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.

A Direct Interaction between the Aryl Hydrocarbon Receptor and Retinoblastoma Protein: LINKING DIOXIN SIGNALING TO THE CELL CYCLE

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In 5L hepatoma cells, TCDD induces a G1 cell cycle arrest through a mechanism that involves the AhR. The retinoblastoma tumor suppressor protein (pRb) controls cell cycle progression through G1 in addition to promoting differentiation. We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. In vivo and in vitro assays reveal a direct interaction between pRb and the AhR but not the AhR nuclear translocator protein. Binding between the AhR and pRb occurs through two distinct regions in the AhR. A high affinity site lies within the N-terminal 364 amino acids of the AhR, whereas a lower affinity binding region colocalizes with the glutamine-rich transactivation domain of the receptor. AhR ligand binding is not required for the pRb interaction per se, although immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation. These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity.

Conservation and divergence of NF-Y transcriptional activation function.

The CCAAT-binding protein NF-Y is involved in the regulation of a variety of eukaryotic genes and is formed in higher eukaryotes by three subunits NF-YA/B/C. We have characterized NF-Y of the trematode parasite Schistosoma mansoni and studied the structure and the function of the SMNF-YA subunit. In this work, we present the cloning and sequence analysis of the B subunit of the parasite factor. SMNF-YB contains the conserved HAP-3 homology domain but the remaining part of the protein was found to be highly divergent from all other species. We demonstrated by transfections of GAL4 fusion constructs, that mouse NF-YB does not contain activation domains while the C-terminal part of SMNF-YB has transcriptional activation potential. On the other hand, the N-terminal parts of SMNF-YA and mouse NF-YA were shown to mediate transactivation; the integrity of a large 160 amino acid glutamine-rich domain of NF-YA was required for this function and an adjacent serine- and threonine-rich domain was necessary for full activity in HepG2, but redundant in other cell types. Transactivation domains identified in SMNF-YB are also rich in serine and threonine residues. Our results indicate that serine/threonine-richsequences from helminth parasites potentiate trans-cription and that such structures have diverged during evolution within the same transcription factor.

Normal myeloid development requires both the glutamine-rich transactivation domain and the PEST region of transcription factor PU.1 but not the potent acidic transactivation domain.

Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore, PU.1(-/-) embryonic stem (ES) cells fail to differentiate into Mac-1(+) and F4/80(+) macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability of PU. 1(-/-) ES cells to differentiate into macrophages. In this study, we take advantage of our PU.1(-/-) ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1(-/-) ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.

The two activation domains of the CCAAT-binding factor CBF interact with the dTAFII110 component of the Drosophila TFIID complex.

The CCAAT-binding factor CBF is a heterotrimeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. Previous studies have shown that CBF contains two transcription activation domains: a glutamine-rich, serine-threonine-rich domain present in the CBF-B subunit and a glutamine-rich domain in the CBF-C subunit. In this study, by using a series of deletion mutations of CBF-B and CBF-C in transcription assay in vitro, we further delineated smaller segments in these domains that were sufficient to support transcriptional activation by CBF. To test whether transcription activation by CBF requires co-activators, we examined the interaction between CBF and dTAF110, a component of the Drosophila TFIID complex. Recent work has demonstrated that glutamine-rich domains of the Sp1 transcription factor interact with dTAF110 and that this interaction has an important role in mediating transcription activation. Here we first demonstrate in a direct interaction assay in vitro that CBF binds dTAF110. By using a yeast two-hybrid system we show that both of the transcription activation domains of CBF interact with dTAF110. A deletion analysis suggests that a segment of CBF-B needed for transcription activation is also involved in interaction with dTAF110. In CBF-C the C-terminal portion of the molecule seems to be needed for these two activities. Our results suggest that TAF110 might represent one of the co-activators that mediate transcriptional activation by CBF.

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.

O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions.

The O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins is dynamic and abundant in the nucleus and cytosol. Several transcription factors, including Sp1, have been shown to contain this modification; however, the functional role of O-GlcNAc in these proteins has not been determined. In this paper we describe the use of the previously characterized glutamine-rich transactivation domain of Sp1 (B-c) as a model to investigate the role of O-GlcNAc in Sp1's transcriptionally relevant protein-to-protein interactions with the TATA-binding-protein-associated factor (TAF110) and holo-Sp1. When the model Sp1 peptide was overexpressed in primate cells, this 97-amino-acid domain of Sp1 was found to contain a dominant O-GlcNAc residue at high stoichiometry, which allowed the mapping and mutagenesis of this glycosylation site. In vitro interaction studies between this segment of Sp1 and Drosophila TAF110 or holo-Sp1 indicate that the O-GlcNAc modification functions to inhibit the largely hydrophobic interactions between these proteins. In HeLa cells, the mutation at the mapped glycosylation site was permissive for transcriptional activation. We propose the hypothesis that the removal of O-GlcNAc from an interaction domain can be a signal for protein association. O-GlcNAc may thereby prevent untimely and ectopic interactions.

Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe.

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.

Activation domain requirements for disruption of Epstein-Barr virus latency by ZEBRA.

Latent infection of B lymphocytes by Epstein-Barr virus (EBV) can be disrupted by expression of the EBV ZEBRA protein. ZEBRA, a transcriptional activator, initiates the EBV lytic cascade by activating viral gene expression. ZEBRA is also indispensable for viral replication and binds directly to the EBV lytic origin of replication. The studies described herein demonstrate that the activation domain. ZEBRA activation can be replaced by a heterologous acidic, proline-rich, or glutamine-rich activation domain. ZEBRA activation domain swap constructs retain ZEBRA's native abilities to activate specific EBV promoters, to disrupt EBV latency, and to stimulate replication at the EBV lytic origin. Additional work, employing sequential and internal deletions of ZEBRA's N-terminal activation domain, indicates that its separate activities are not attributable to specific subdomains but are spread throughout its N terminus and therefore cannot be inactivated by deleting localized regions.

Characterization of the Aromatic Hydrocarbon Receptor Gene and Its Expression in Atlantic Tomcod

Cytochrome P4501A1 (CYP1A1) mRNA is not inducible in Atlantic tomcod from the Hudson River that are treated with halogenated aromatic hydrocarbons (HAHs). In contrast, CYP1A1 mRNA is inducible in Hudson River tomcod that are treated with polycyclic aromatic hydrocarbons (PAHs) and in tomcod that are collected from cleaner rivers and treated with HAHs or PAHs. We hypothesize that CYP1A1 transcription is inhibited in Hudson River tomcod because of down-regulation of the aromatic hydrocarbon receptor (AhR) pathway and that separate molecular pathways modulate CYP1A1 transcription in fish treated with HAHs and PAHs. We initially evaluated levels of hepatic nuclear protein binding at enhancer elements (DREs) in the regulatory region of tomcod CYP1A1. No difference in levels of protein binding was observed between tomcod from the Hudson and Miramichi (cleaner) rivers that were untreated or were treated with benzo[a]pyrene. In contrast, levels of protein binding were lower in tomcod from the Hudson River that were treated with TCB than in similarly treated fish from the Miramichi River, suggesting differences between the populations in the structure or expression of AhR pathway molecules. To address this possibility, AhR DNA sequences were characterized from tomcod cDNA and genomic DNA libraries. In tomcod and mammals, AhR is represented by 11 exons, overall peptide sizes are similar, and amino acid sequences at basic, helix–loop–helix, PAAS A, and PAAS B domains are highly conserved. In contrast, little similarity was observed between tomcod and mammals in the sizes or sequences of AhR exons 10 and 11, including the absence in tomcod of glutamine-rich domains. No differences in levels of hepatic AhR mRNA were observed between the two populations or treatment groups when tomcod were untreated or were treated with aromatic hydrocarbons. In contrast, variation in levels of AhR mRNA expression was observed among tomcod tissues; however, no relationship was observed between levels of AhR mRNAs and CYP1A1 mRNAs in tissues from chemically or vehicle control-treated fish. RFLP analysis revealed extensive variation in exons 10 and 11 of AhR cDNA among tomcod from different rivers. Our results suggest that variation between tomcod populations in CYP1A1 mRNA inducibility is reflected by differences in levels of inducible hepatic protein binding to DREs. However, levels of hepatic AhR mRNA are not down-regulated in the Hudson River population, are not affected by AH treatments, and levels of AhR mRNA expression are not responsible for the differential inducibility of CYP1A1 transcription.

Control of germ-band retraction in Drosophila by the zinc-finger protein HINDSIGHT.

Drosophila embryos lacking hindsight gene function have a normal body plan and undergo normal germ-band extension. However, they fail to retract their germ bands. hindsight encodes a large nuclear protein of 1920 amino acids that contains fourteen C2H2-type zinc fingers, and glutamine-rich and proline-rich domains, suggesting that it functions as a transcription factor. Initial embryonic expression of hindsight RNA and protein occurs in the endoderm (midgut) and extraembryonic membrane (amnioserosa) prior to germ-band extension and continues in these tissues beyond the completion of germ-band retraction. Expression also occurs in the developing tracheal system, central and peripheral nervous systems, and the ureter of the Malpighian tubules. Strikingly, hindsight is not expressed in the epidermal ectoderm which is the tissue that undergoes the cell shape changes and movements during germ-band retraction. The embryonic midgut can be eliminated without affecting germ-band retraction. However, elimination of the amnioserosa results in the failure of germ-band retraction, implicating amnioserosal expression of hindsight as crucial for this process. Ubiquitous expression of hindsight in the early embryo rescues germ-band retraction without producing dominant gain-of-function defects, suggesting that hindsight's role in germ-band retraction is permissive rather than instructive. Previous analyses have shown that hindsight is required for maintenance of the differentiated amnioserosa (Frank, L. C. and Rushlow, C. (1996) Development 122, 1343-1352). Two classes of models are consistent with the present data. First, hindsight's function in germ-band retraction may be limited to maintenance of the amnioserosa which then plays a physical role in the retraction process through contact with cells of the epidermal ectoderm. Second, hindsight might function both to maintain the amnioserosa and to regulate chemical signaling from the amnioserosa to the epidermal ectoderm, thus coordinating the cell shape changes and movements that drive germ-band retraction.

Alternative system for detection and mapping of activation domains.

The ZEBRA protein is a transcriptional activator that induces expression of viral lytic genes in cells harboring latent Epstein-Barr virus (EBV). In this report it is shown that a derivative of ZEBRA that cannot activate transcription (Zd) can be used to detect and characterize activation domains. Three expression vectors that allow the fusion of putative activation regions in any reading frame were constructed using Zd. These vectors were used to demonstrate the activity of different classes of activation domains using a chloramphenicol acetyltransferase (cat) reporter gene construct containing seven ZEBRA response elements (Z7). The Zd/Z7 system effectively detected proline-rich, glutamine-rich and acidic activation domains in a variety of cell lines and cell types. Using a bioassay unique to the EBV Zd/Z7 system, fusion constructs can also be tested for the ability to activate gene expression directly from a chromatin structure, the EBV genome. These studies indicate that the Zd/Z7 system is an alternative to GAL4 and can be a useful tool for identifying heterologous activation domains.

The ability of C/EBP beta but not C/EBP alpha to synergize with an Sp1 protein is specified by the leucine zipper and activation domain.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBP alpha and C/EBP beta are expressed abundantly in liver, only C/EBP beta is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP beta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP beta proteins containing either the leucine zipper or the activation domain of C/EBP alpha are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP beta are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP beta. Furthermore, analysis of c/ebp beta-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP beta to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.