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

No description available.

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.

No description available.

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.

Retinoic acid (RA), the major biologically active form of vitamin A, plays important roles in regulating a broad range of biological processes. / Progressive loss of RARs is associated with skin carcinogenesis both in human and animals. Despite such observations, the biological significance of RAR loss in skin carcinogenesis has not yet been clarified. To this end, we established keratinocyte cell lines deficient in RARalpha, RARgamma, or both and employed a well-established tumorigenesis model to investigate whether loss of RARs is causally related to skin tumorigenesis. We found that RARgamma is the major RAR subtype mediating the growth and AP-1 inhibitory effects of RA on keratinocytes in vitro. Consistent with this observation, loss of RARgamma, but not RARalpha, predisposed keratinocytes to tumor formation, suggesting that RARgamma may act as a tumor suppressor. Reconstitution of RARs in the RARalphagamma-/- keratinocytes inhibited their tumorigenic potential, further proving that RARs have tumor suppressive effects. / As expected, expression of dnRARalpha resulted in profound epidermal defects. Intriguingly, dnRAalphaDBD caused a virtually identical skin phenotype, suggesting that dnRARalpha acts to affect epidermal development via a DNA-binding-independent mechanism. The epidermal phenotype of these transgenic mice is reminiscent of that seen in the p63-/- mice, and p63 expression was indeed significantly reduced in the epidermis expressing dnRARalpha or dnRARalphaDBD, suggesting that downregulation of p63 by dnRARalpha may be attributable to the epidermal phenotypes associated with the transgenic mice. These observations also suggest that DNA-binding is not required for dnRARalpha to attenuate p63 expression in the epidermis. Consistent with these observations, I also found that p63 is indeed not a RAR-target, as no overt changes in p63 expression were observed in the RARalphagamma-/- epidermis, which appeared normal. (Abstract shortened by UMI.)

Acute Promyelocytic Leukemia (APL) results from an accumulation of undifferentiated promyelocyte progenitors. Complete clinical remission of APL can be achieved through therapy with retinoic acid (RA). However, patients treated with RA alone almost invariably develop resistance to RA. We have found that tumor necrosis factor (TNF) and RA can synergize to induce differentiation of RA sensitive APL cells in a much shorter period of time than RA alone. In addition, this combination can also overcome the maturation block of RA resistant APL cells and the non-APL leukemia cell line U937 leading to differentiation. Correlating with this, we found synergistic induction of several genes at early and late time points in both the RA sensitive and resistant cell lines. Through use of neutralization antibodies, we found the protein product of one of these genes, the receptor for the macrophage colony stimulating factor, was important in mediating the differentiation effects of TNF and RA. / To better understand transcriptional activation with RA and TNF we used the promoter of a gene synergistically induced by RA and TNF, Dif-2, as a model to investigate the mechanism whereby the two agents interact on the level of transcription at early time periods. We used ChIP analysis to study the accessibility of the promoter to binding by various transcription factors and the recruitment of cofactors in response to RA and TNF. We found that upon RA treatment, there was a release of corepressors and a decrease in histone methylation. This was accompanied by a subsequent increase in binding of the transcription factor PU.1, a recruitment of coactivators, as well as Snf5 (a component of the Swi/Snf complex), and an increase in histone acetylation. Interestingly, TNF could only stimulate NF-kappaB (downstream effectors of TNF) binding to the Dif-2 promoter when cells were cotreated with RA. Furthermore, TNF and RA lead to a heightened level of active, phosphorylated RNA Pol II at the Dif-2 promoter. Correlating with this was heightened recruitment of TFIIH, a known Pol II kinase. This may represent a mechanism whereby RA and TNF act in synergy to activate Pol II. These data suggest a novel mechanism for synergy between signaling pathways where RA can trigger a conformation change in a target promoter/enhancer region resulting in a more open conformation that is conducive to transcription factor binding in response to other stimuli.