FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis.

Orphan receptors of the FTZ-F1-related group of nuclear receptors (xFF1r) were identified in Xenopus laevis by isolation of cDNAs from a neurula stage library. Two cDNAs were found, which encode full length, highly related receptor proteins, xFF1rA and B, whose closet relative known so far is the murine LRH-1 orphan receptor. xFF1rA protein expressed by a recombinant vaccinia virus system specifically binds to FTZ-F1 response elements (FRE; PyCAAGGPyCPu). In cotransfection studies, xFF1rA constitutively activates transcription, in a manner dependent on the number of FREs. The amounts of at least four mRNAs encoding full-length receptors greatly increase between gastrula and early tailbud stages and decrease at later stages. At early tailbud stages, xFTZ-F1-related antigens are found in all nuclei of the embryo.

Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

Pro-inflammatory cytokines induce the transcription factors C/EBPbeta and C/EBPdelta in astrocytes.

The transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and -delta are key regulators for the expression of the acute phase genes in the liver, such as complement component C3 and antichymotrypsin. In the brain, these acute phase proteins are produced in response to pro-inflammatory cytokines by the reactive astrocytes, in particular those surrounding the amyloid plaques of Alzheimer's disease brains. Here we show that lipopolysaccharides (LPS), IL-1beta, and TNFalpha induce the expression of the c/ebpbeta and -delta genes in mouse primary astrocytes. This induction precedes the expression of the acute phase genes coding for the complement component C3 and the mouse homologue of antichymotrypsin. The induction of these two acute phase genes by LPS is blocked by cycloheximide, whereas this protein synthesis inhibitor does not affect the expression of the c/ebp genes. Altogether, our data support a role as immediate-early genes for c/ebpbeta and -delta, whose expression is induced by pro-inflammatory cytokines in mouse cortical astrocytes. In the liver, these transcription factors are known to play an important role in inflammation and energy metabolism regulation. Therefore, C/EBPbeta and -delta could be pivotal transcription factors involved in brain inflammation, in addition to their previously demonstrated role in brain glycogen metabolism regulation (Cardinaux and Magistretti. J Neurosci 16:919-929, 1996).

Growth promoting signaling by tenascin-C [corrected].

Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of beta-catenin and stimulated the expression of the beta-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Role of penicillin-binding proteins in Streptococcus gordonii

Résumé Streptococcus gordonii est une bactérie colonisatrice naturelle de la cavité buccale de l'homme. Bien que normalement commensale, elle peut causer des infections graves, telles que des bactériémies ou des endocardites infectieuses. La pénicilline étant un des traitements privilégiés dans de tels cas, l'augmentation rapide et globale des résistances à cet antibiotique devient inquiétante. L'étude de la physiologie et des bases génétiques de ces résistances chez S. gordonii s'avère donc importante. Les cibles moléculaires privilégiées de la pénicilline G et des β-lactames sont les penicilllin-binding proteins (PBPs). Ces enzymes associées à la membrane ont pour rôle de catalyser les réactions de transpeptidation et de transglycosylation, qui constituent les dernières étapes de la biosynthèse du peptidoglycan (PG). Elles sont définies comme classe A ou B selon leur capacité d'assurer soit les deux réactions, soit uniquement la transpeptidation. Les β-lactames inhibent le domaine transpeptidase de toutes les PBPs, entraînant l'inhibition de la synthèse du PG, l'inhibition de la croissance, et finalement la mort cellulaire. Chez les streptocoques, les PBPs sont aussi les premiers déterminants de la résistance à la pénicilline. De plus, elles sont impliquées dans la morphologie bactérienne, en raison de leur rôle crucial dans la formation du PG. Le but de ce travail était de caractériser les PBPs de S. gordonii et d'étudier leurs fonctions dans la vie végétative de la bactérie ainsi que durant le développement de la résistance à la pénicilline. Premièrement, des mutants auxquels il manque une ou deux PBP(s) ont été construits. Leur étude - au niveau physiologique, biochimique et morphologique - a montré le caractère essentiel ou dispensable de chaque protéine, ainsi que certaines de leurs fonctions potentielles. Deuxièmement, des mutants résistants à la pénicilline ont été générés. Leur caractérisation a montré l'importance des mutations dans les PBPs ainsi que dans d'autres gènes encore inconnus, de même que le rôle crucial des PBPs de classe A dans le développement de la résistance à la pénicilline. Des expériences supplémentaires sur des isolats résistants ont aussi prouvé que la résistance a un coût en terme de fitness, coût que S. gordonii parvient à compenser par des mécanismes d'adaptation. Finalement, les promoteurs des gènes des PBPs ont été déterminés et leur expression a été étudiée grâce au gène de luciférase. Il a ainsi été montré que la résistance à la pénicilline entraîne non seulement des altérations au niveau des protéines, mais aussi au niveau de la régulation des gènes. De plus, la pénicilline génère directement des modifications dans l'expression de PBPs spécifiques. Summary Streptococcus gordonii is a normal inhabitant of the human oral cavity and a pioneer colonizer of teeth. Although usually considered as a commensal, this organism can cause life-threatening infections such as bacteraemia or endocarditis. Since penicillin is one of the preferential treatments for such pathologies, the rapid and general increase of antibiotic resistance in the overall population becomes an issue. Thus, studying the physiologic and genetic bases of such a resistance in S. gordonii is of interest. The primary molecular targets of penicillin G and other β-lactams are the so called penicillin-binding proteins (PBPs). These are membrane-associated proteins that catalyze the last steps in peptidoglycan (PG) biosynthesis, namely transpeptidation and transglycosylation. Depending on their capacity to catalyze either reactions or only transpeptidation, they are considered as class A or class B PBPs, respectively. β-lactam antibiotics inhibit the transpeptidase domain of both of these classes of enzymes, resulting in inhibition of PG assembly, inhibition of bacterial growth, and ultimately leading to cell death. In streptococci, PBPs are also the primary determinants of penicillin-resistance. Moreover, because of their crucial role in PG formation, they are implicated in fundamental aspects of cell morphology. The goal of this work was thus to characterize S. gordonii PBPs and to explore their functions in terms of vegetative life and penicillin-resistance development. First, single and double PBP-inactivated mutants were generated and their effect on the bacterial physiology, cell wall biochemistry and ultrastructural morphology was assessed. This demonstrated the essentiality or dispensability of each protein for bacterial life. Second, penicillin-resistant mutants were generated by cyclic exposure to increasing concentrations of the drug. Characterization of these mutants pointed out the importance of both PBP and non-PBP mutations, as well as the crucial role of the class A PBPs in the development of penicillin-resistance. Further experiments on resistant isolates demonstrated the fitness cost of this resistance, but also the capacity of S. gordonii to adapt and regain the fitness of the wild-type. Finally, the promoters of PBP genes were determined and their expression was monitored using luciferase fusions. This showed that penicillin-resistance, in addition to modifications at the level of the protein, also triggered genetic alterations. Moreover, penicillin itself generated modifications in the expression of specific PBPs.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

The Xenopus vitellogenin (vit) gene B1 estrogen-inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen-responsive elements (EREs), i.e. ERE-2 and ERE-1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor- and hormone-dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen-responsive unit (vitB1 ERU). Using in vitro protein-DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE-2 increases approximately 4- to 8-fold the binding affinity of the receptor to the adjacent less conserved ERE-1. Thus, we suggest that the observed synergistic estrogen-dependent transcription activation conferred by the pair of hormone-responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants.

The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B-TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein-protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.

Viral transport of DNA damage that mimics a stalled replication fork.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

Stereospecific recognition of pyochelin and enantio-pyochelin by the PchR proteins in fluorescent pseudomonads.

The siderophore pyochelin of Pseudomonas aeruginosa promotes growth under iron limitation and induces the expression of its biosynthesis genes via the transcriptional AraC/XylS-type regulator PchR. Pseudomonas fluorescens strain CHA0 makes the optical antipode of pyochelin termed enantio-pyochelin, which also promotes growth and induces the expression of its biosynthesis genes when iron is scarce. Growth promotion and signalling by pyochelin and enantio-pyochelin are highly stereospecific and are known to involve the pyochelin and enantio-pyochelin outer-membrane receptors FptA and FetA, respectively. Here we show that stereospecificity in signalling is also based on the stereospecificity of the homologous PchR proteins of P. aeruginosa and P. fluorescens towards their respective siderophore effectors. We found that PchR functioned in the heterologous species only if supplied with its native ligand and that the FptA and FetA receptors enhanced the efficiency of signalling. By constructing and expressing hybrid and truncated PchR regulators we showed that the weakly conserved N-terminal domain of PchR is responsible for siderophore specificity. Thus, both uptake and transcriptional regulation confer stereospecificity to pyochelin and enantio-pyochelin biosynthesis.

Mutational analysis of class A and class B penicillin-binding proteins in Streptococcus gordonii.

High-molecular-weight (HMW) penicillin-binding proteins (PBPs) are divided into class A and class B PBPs, which are bifunctional transpeptidases/transglycosylases and monofunctional transpeptidases, respectively. We determined the sequences for the HMW PBP genes of Streptococcus gordonii, a gingivo-dental commensal related to Streptococcus pneumoniae. Five HMW PBPs were identified, including three class A (PBPs 1A, 1B, and 2A) and two class B (PBPs 2B and 2X) PBPs, by homology with those of S. pneumoniae and by radiolabeling with [3H]penicillin. Single and double deletions of each of them were achieved by allelic replacement. All could be deleted, except for PBP 2X, which was essential. Morphological alterations occurred after deletion of PBP 1A (lozenge shape), PBP 2A (separation defect and chaining), and PBP 2B (aberrant septation and premature lysis) but not PBP 1B. The muropeptide cross-link patterns remained similar in all strains, indicating that cross-linkage for one missing PBP could be replaced by others. However, PBP 1A mutants presented shorter glycan chains (by 30%) and a relative decrease (25%) in one monomer stem peptide. Growth rate and viability under aeration, hyperosmolarity, and penicillin exposure were affected primarily in PBP 2B-deleted mutants. In contrast, chain-forming PBP 2A-deleted mutants withstood better aeration, probably because they formed clusters that impaired oxygen diffusion. Double deletion could be generated with any PBP combination and resulted in more-altered mutants. Thus, single deletion of four of the five HMW genes had a detectable effect on the bacterial morphology and/or physiology, and only PBP 1B seemed redundant a priori.

One billion years of bZIP transcription factor evolution: conservation and change in dimerization and DNA-binding site specificity.

The genomic era has revealed that the large repertoire of observed animal phenotypes is dependent on changes in the expression patterns of a finite number of genes, which are mediated by a plethora of transcription factors (TFs) with distinct specificities. The dimerization of TFs can also increase the complexity of a genetic regulatory network manifold, by combining a small number of monomers into dimers with distinct functions. Therefore, studying the evolution of these dimerizing TFs is vital for understanding how complexity increased during animal evolution. We focus on the second largest family of dimerizing TFs, the basic-region leucine zipper (bZIP), and infer when it expanded and how bZIP DNA-binding and dimerization functions evolved during the major phases of animal evolution. Specifically, we classify the metazoan bZIPs into 19 families and confirm the ancient nature of at least 13 of these families, predating the split of the cnidaria. We observe fixation of a core dimerization network in the last common ancestor of protostomes-deuterostomes. This was followed by an expansion of the number of proteins in the network, but no major dimerization changes in interaction partners, during the emergence of vertebrates. In conclusion, the bZIPs are an excellent model with which to understand how DNA binding and protein interactions of TFs evolved during animal evolution.

Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.