The effects of productive adenovirus infection on host gene expression were studied using a line of methotrexate-resistant HeLa cells containing amplified amounts of the gene coding for dihydrofolate reductase (DHFR). These cells contain several hundred copies of the DHFR gene and overproduce DHFR RNA and protein. Synthesis of DHFR is induced early in adenovirus infection, before the onset of viral replication, and is suppressed late in infection along with general host protein synthesis. These changes in DHFR protein synthesis are accompanied by changes in both the steady state cytoplasmic levels of DHFR mRNA and the rate of appearance of DHFR mRNA into the cytoplasm. These data indicate that nuclear events are important in the control of DHFR gene expression during productive infection. Transcription of DHFR-specific sequences continues at a constant rate throughout infection. In contrast, steady state nuclear levels of DHFR-specific sequences change in correspondence to the changing rate of appearance of DHFR mRNA into the cytoplasm through the course of infection. Thus, constitutive transcription of DHFR sequences is followed by a differential accumultion of DHFR nuclear RNA at different times during infection, indicating that post-transcriptional events are important in the regulation of DHFR gene expression by adenovirus. Sepcifically, the regulation of DHFR gene expression during infection is accomplished by a modulation of the relative stability of DHFR-specific sequences through the course of infection. During induction, DHFR sequences are more stable than in either uninfected cells or infected cells late in infection. The induction of DHFR is temporally linked to viral replication during both wild type infection and infection with defined early adenoviral mutants which delay replication. Studies with mutant defective in the production of E4 or E1b gene products indicate that E4 gene products are involved in the switch from early to late viral gene expression during the time of DHFR induction, and that both E4 and E1b gene products are necessary for the efficient shut off of DHFR and other host genes late in infection.

To determine the mechanisms that the yeast S. cerevisiae uses to adapt to osmotic stress, mutants were isolated which were impaired in their ability to accumulate glycerol in response to a high osmolarity challenge. These mutants identified four genes, HOG1 through HOG4 (High Osmolarity Glycerol Response). HOG2 was cloned from a genomic library on a centromere vector by complementation of the high osmolarity sensitive growth (Osm$\sp{\rm s}$) phenotype of hog2-1. Integrative confirmation of the genetic linkage between the cloned DNA and the locus of the original point mutation was performed. The null mutant hog2-$\Delta$3, created by deletion of the gene, had phenotypes similar to those of hog2-1. In addition to being Osm$\sp{\rm s}$, hog2-$\Delta$3 mutants are heat shock-sensitive, fail to accumulate glycogen and are defective for sporulation. We sequenced HOG2 and determined that this gene encodes a 102 kDa protein, the N-terminus of which contains a region of 350 amino acids showing 38% identity to CIF1(GLC6), a gene required for metabolic responses to glucose. HOG2 encodes trehalose-6-phosphate phosphatase. HOG2 mRNA levels increase in response to heat shock and osmotic stress. Yeast cells containing a HOG2::lacZ fusion showed a four-fold increase in $\beta$-galactosidase one hour after exposure to 0.5 M NaCl, but no increase was seen after a heat shock at 42$\sp\circ$ for one hour. The pleiotropic phenotype of hog2$\Delta$ mutants suggests that HOG2 is a regulatory gene necessary for adaptive responses to a variety of environmental stresses. We propose that the promoter region of HOG2 contains two separate stress elements in the Upstream Activating Sequence (UAS). We suggest that the HOG2 promoter region located between nucleotides $-$1 and $-$400 of the HOG2 coding region contains a novel osmotic stress element which is induced in response to an increased extracellular osmolarity. The inducement of this osmotic stress element is not dependent on the presence of the TRS, the heat shock inducible element, found farther upstream.

The biodegradative arginine decarboxylase and lysine decarboxylases, encoded by adi and cadA, respectively, are induced to maximal levels when E. coli is grown anaerobically in rich medium at acidic pH. They were used as model systems to study how E. coli responds to a specific environmental stress, moderately acidic pH. Two Mu dlac fusion strains, GNB7145K (adi::lac) and GNB8385K (cadA::lac), were used as genetic tools in our search for transregulating factors involved in acid-induction of these two gene systems. By characterization of random transposon mutagenesis-generated mutants that showed altered expression patterns in response to medium pH, and by studying a series of plasmids that could complement the mutant phenotypes, we identified six genes whose products might be involved in the acid-induction processes. Four of them are likely to be responsible for the basal level of expression at noninducing conditions, three of which, hns, hnsB, and hfq, code for known or putative bacterial histone-like proteins, and one of which, leuO, codes for a putative transcription regulator. The other two, rpoA encoding the $\alpha$ subunit of RNA polymerase, and cysB encoding the positive regulator for cysteine biosynthesis operons, are likely to be involved in the adi activation process at inducing conditions. These findings have allowed us to speculate upon the mechanisms of acid-regulation, particularly of the adi gene system where the regulation seems complex and little previous information was available.

The core domain (residues 62-323) of the regulatory protein lac repressor has been aligned to several sugar binding proteins of known structure. The overall homology based on two separate matrix scoring systems (minimum base change per codon and amino acid homology per residue) is significant. Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of lac repressor was modeled using the structures of the sugar binding proteins as templates. Further refinements of this model using the purine repressor provided improvement in regions not well defined based on the sugar binding proteins. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. The recent solution of the crystallographic structure confirms the elements of this homology model. This approach provides an effective means to visualize the core domain of the lac repressor and to interpret the mutational data for specific residues. The availability of models of this type provides a structural basis for rational design of experiments. Mutations that affect both monomer-monomer and dimer-dimer subunit interfaces have been combined to generate a new family of mutant proteins designed to explore the role of subunit interactions in this regulatory protein. Combination of apolar substitutions at residue 84 and C-terminal deletions to generate dimer results in mutant repressor proteins with increased stability, even in the presence of Tyr282Asp, a mutation that generates monomer in the wild-type background. Operator binding studies demonstrate the linkage between dimer formation and operator binding and confirm that dimers are the minimal unit required for repression. The nature of the side chain at position 84 not only influences subunit association and stability, but also appears to contribute to subunit communication and DNA binding domain orientation. Placement of the sites of these mutations in the homology model of the lac repressor is consistent with the characteristics found for these proteins.

Adaptation of plants to the changing environment requires that sensing of external stimuli be linked to the mechanisms of morphogenesis. The Arabidopsis TCH genes are rapidly and strongly regulated in expression by a variety of environmental stimuli. The physiological relevance of TCH gene induction is not clear. We identified TCH4 as a xyloglucan endotransglycosylase (XET) by sequence similarity and enzyme activity. XET modifies xyloglucan, an important component of dicots cell wall, which is a fundamental determinant of cell shape and plant form. Therefore TCH4 product is very likely to affect the cell wall mechanical properties, and the magnitude and direction of cell expansion. The expression pattern of TCH4-GUS fusion gene and the pattern of endogenous TCH4 protein localization revealed that TCH4 is likely expressed in cells that are undergoing cell extension and cells undergoing cell wall modification. In addition to the developmental regulation, the expression of TCH4 is upregulated in plants subjected to environmental stimuli such as touch, darkness, heat shock, cold shock, and plant growth regulators, auxin and brassinosteroid. The function of TCH4 as an XET and its unique regulation property indicate that TCH4 may underlie plant morphogenetic responses to the environmental cues and to the endogenous hormones. A 1 kb TCH4 region was determined to be able to confer the inducibility of expression to the GUS reporter gene for all stimuli tested. Further dissection of the 1 kb TCH4 sequences revealed that the cis-acting elements conferring the inducibility in response to different stimuli are separable. The TCH4 regions conferring TCH4 tissue-specific expression was also determined. The TCH4 expression regulated by environmental, hormonal and developmental factors is regulated through a complex collection of cis-regulatory elements and may occurs through both transcriptional and post-transcriptional mechanisms. Genomic Southern analysis indicates that there is a TCH4-related gene family in Arabidopsis. These genes encode unique XET-related proteins and show differential regulation by environmental and hormonal stimuli. Elucidation of the functions and regulation of this gene family will most likely lead to an insight into the role of the plant cell wall-modifying enzymes in the morphogenesis of plant growth and development responding to the environment.

Overexpression of the ErbB2 receptor tyrosine kinase is observed in 20-30% of human breast cancers and correlates with poor prognostic outcome. To identify novel drug targets to treat ErbB2 positive breast cancer, a better understanding of both the processes regulating ErbB2 overexpression and the oncogenic/metastatic signals downstream of ErbB2 is required. We had previously identified EGR2 and CITED1 as transcription factors upregulated in mammary tumors from mice expressing activated ErbB2 from the endogenous erbB2 promoter compared to those expressing activated ErbB2 from the MMTV promoter. Here we validate EGR2 as an erbB2 transcriptional regulator which can activate expression from the erbB2 promoter. We also demonstrate that EGR2 associates with the CITED1 coactivator which results in enhanced EGR2-mediated transcriptional activation. Furthermore, 14-3-3sigma also associates with EGR2 which results in the cytoplasmic relocalization of EGR2, suggesting one mechanism by which this putative tumor suppressor may inhibit tumorigenesis. An important group of signaling molecules downstream of activated ErbB2 is the Akt family of kinases. We have previously shown that Akt1 can accelerate mammary tumorigenesis in ErbB2 and PI3K-uncoupled PyVmT transgenic mice. Therefore we generated transgenic mice expressing Akt2 in the mammary epithelium to examine the role of this isoform. Interbreeding Akt2 mice with ErbB2 and PI3K-uncoupled PyVmT transgenics did not affect tumor latency, but instead increased metastasis in both models. Furthermore, downregulation of endogenous Akt2 in highly metastatic ErbB2 mammary tumor-derived cell lines decreased invasion. In the ErbB2-based tumors, both Akt1 and Akt2 expression altered the levels of EGFR family members. Moreover, Akt1 expressing tumors displayed increased ERalpha, whereas Akt2 expressing tumors displayed decreased p-p38 MAPK. To more thoroughly examine the differ / Le récepteur tyrosine kinase ErbB2 est surexprimé dans 20 à 30 % des cancers du sein, cette surexpression étant corrélée à un mauvais pronostic. Dans le but d'identifier de nouvelles cibles pharmacologiques permettant de traiter les cancers du sein ErbB2-positifs, il est donc nécessaire d'acquérir une meilleure compréhension d'une part, des processus induisant la surexpression de ErbB2, et d'autre part, des voies de signalisation couplées et impliquées dans le potentiel oncogénique et métastatique de ce récepteur. De précédents travaux nous avaient permis d'identifier EGR2 et CITED1 comme facteurs de transcription dont l'expression est augmentée dans les tumeurs mammaires développées chez les souris exprimant une forme activée de ErbB2 lorsqu'elle est sous la dépendance du promoteur endogène de erbB2 (et non pas lorsqu'elle est sous la dépendance du promoteur MMTV). Les travaux présentés ici nous permettent de valider EGR2 comme étant un authentique régulateur transcriptionnel de erbB2 et permettant d'activer une expression dépendante du promoteur de erbB2. Nous démontrons également qu'EGR2 s'associe au co-activateur CITED1 induisant une augmentation de l'activation transcriptionnelle induite par EGR2. De plus, le suppresseur de tumeur putatif 14-3-3sigma est aussi capable de s'associer à EGR2 entraînant sa relocalisation cytoplasmique et suggérant ainsi un mécanisme par lequel 14-3-3sigma pourrait inhiber la tumorigénèse.La famille de kinases Akt constitue un groupe important de molécules du signal intracellulaire couplé à la forme activée de ErbB2. Notre groupe ayant précédemment démontré qu'Akt1 était capable d'accélérer la tumorigénèse mammaire chez des souris transgéniques exprimant ErbB2 ou un mutant de PyVmT découplé de la PI3K, nous avons généré des souris transgénique exprimant Akt2 au niveau de l'épithélium mammaire afin d'étudier le r

Proteins of the Ner/Nlp family have been characterized in order to identify the evolutionarily conserved protein domains important to their structure and function. The homologous DNA-bonding Ner proteins of closely related temperate coliphages Mu and D108 regulate the transcription and transposition of the phage genomes by binding to nonhomologous DNA operators. In order to precisely define the DNA-binding domains of Mu and D108 Ner, recombinant Mu and D108 ner genes were constructed and the DNA-binding and pseudoimmunity properties of the recombinant proteins were examined. / D3112 is a Mu-like transposable bacteriophage of Pseudomonas aeruginosa which was proposed to encode a ner-like gene termed cip. In order to determine if cip is equivalent to ner, the D3112 left end was sequenced. Six possible open reading frames (ORFs) for Cip were identified, yet none were found to be biochemically homologous to Ner. However, an E. coli gene (nlp) which encodes a Ner-like protein (Nlp) has recently been identified. This gene product, when overexpressed in a cya crp*l strain, stimulates expression of the mal operon. We have demonstrated that nlp is not essential for E. coli viability and stability. The nlp gene is transcribed in E. coli and sequences homologous to nlp have been identified in the Enterobacteriaceae, but not in Pseudomonas aeruginosa. These studies indicate that Nlp, like its Ner counterparts, may play an important regulatory role and that these functions may be mediated by conserved protein structures.

The tumor suppressor gene RB regulates cell proliferation at the G1/S transition of the cell cycle. The retinoblastoma protein pRB associates with both HDAC-dependent and independent mechanisms to actively repress E2F-dependent genes required for entry into S phase. The retinoblastoma binding protein 1 RBP1 recruits the mSin3A/HDAC1 co-repressor complex to the pocket of pRB at growth arrest and accounts for the majority of the HDAC activity associated with pRB. However, transcriptional repression by RBP1 also involves HDAC-independent activities because repression is only partially relieved by the HDAC inhibitor Trichostatin A. This activity is mediated in part by residues 241 to 452 of RBP1 designated as the R1 domain. Hypermapping studies on the previously defined R1 domain of RBP1 revealed that amino acids 400 to 452 of RB1 are sufficient to mediate HDAC-independent repression. Inspection of the minimal R1 region located two copies of the SUMO consensus motif Psi-K-X-E and subsequent experiments demonstrated that the R1 domain is post-translationally modified by SUMO on lysine 418 and 444. In addition, transcriptional repression by the R1 domain was abrogated by either mutagenesis of both SUMO acceptor lysines or in the presence of a SUMO specific protease implying that SUMO modification modulates HDAC-independent transcriptional repression by RBP1.

Regulation of chromatin structure involves histone modifications such as acetylation. Since 1996, the identification and characterization of histone acetyltransferases have had tremendous impact on our understanding of the molecular mechanisms related to eukaryotic gene regulation and human diseases associated with abnormal chromatin functions. The MYST family of histone acetyltransferases is very interesting because of their various biological functions. In agreement with the correlation between aberrant histone acetylation and cancer, the MYST family proteins MOZ and MORF are linked to leukemogenesis. / Identification and characterization of a gene encoding a novel histone acetyltransferase were the goals of this thesis project. Human MORF gene was cloned and the encoded protein, MORF, was shown to be very similar to MOZ. Biochemical studies demonstrated that both MOZ and MORF possess intrinsic histone acetyltransferase activities. The amino- and carboxy-terminal regions of MOZ and MORF contain transcriptional repression and activation domains, respectively. / Runx2, an osteoblast-specific transcription factor, binds to the activation domains of MOZ and MORF and thus recruits them to the osteocalcin promoter for transcriptional activation. TAZ, a WW-domain transcriptional coactivator of Runx2, potentiates the transcriptional activation of the osteocalcin promoter by MOZ and Runx2. Interestingly, treatment of cells with PMA enhances the synergy between MOZ and TAZ in activating the osteocalcin promoter. Consistent with this, PMA treatment strengthens the interaction of Runx2 with MOZ and TAZ. / This study, therefore, identified the histone acetyltransferase MORF and demonstrated that MOZ and MORF are transcriptional coactivators, thus providing new insights into how histone acetyltransferases are implicated in cell differentiation and leukemogenesis.

T cell receptor (TCR) engagement triggers a series of biochemical and signaling cascades including protein tyrosine kinase (PTK) activation, tyrosine phosphorylation of adapter proteins and multiple protein-protein interactions. CD45 plays a critical role in regulating TCR-mediated signaling. Here, we report evidence of in vivo interaction between CD45 and the Src kinase-associated phosphoprotein (SKAP55), which acts as its substrate for dephosphorylation. Further, we demonstrate and confirm by mutational analysis that a critical residue of SKAP55, Tyr-232, mediated the association with CD45. In Jurkat cells, SKAP55 induced tyrosine phosphorylation by anti-CD3 stimulation. Biochemical analysis revealed that adapter protein SKAP55 formed homodimers through its SH3 domain and SK region. The amount of SKAP55 homodimer was enhanced in T cell activation induced by anti-CD3 stimulation. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase was dependent on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused MAP kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T-cells. Mutational analysis revealed that Tyr-271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate MAP kinase activation. / Intriguingly, TCR engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts, while Fyn kinase, which was observed in lipid rafts in resting T cells, was found in increased amounts upon TCR activation. The association between SKAP55 and Fyn kinase was also found in lipid rafts, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation. Overexpression of SKAP55 in these cells induced transcriptional activation of the IL-2 promoter, while total suppression of the IL-2 promoter activity was observed for the SKAP55-Y232F mutant. Furthermore, overexpression of SKAP55-Y232F also caused the tyrosine-hyperphosphorylation of Fyn with a decreased kinase activity. Thus, SKAP55 is an essential adaptor which couples CD45 with the Src family kinase Fyn for dephosphorylation, and acts thereby as a key component for positive regulation in TCR signaling.