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Workshop E
Cell Cycle and Control of Transcription (Part 1)
Jakob Troppmair, Würzburg
Ralf Hass, Hannover
Overview
Cell Cycle and Control of Transcription
Michael Naumann
(see also P56)
Coactivators in the transcriptional regulation by STAT5 and STAT6
Litterst C., Marilley D., Gingras S., Groner B., Simard J., Pfitzner E. Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Str. 42-44, Phone: 069/63395116, Fax: 069/63395297, eMail: Litterst@em.uni-frankfurt.de STAT (signal transducer and activators of transcription) are central components in the signal transduction events in hematopoietic and epithelial cells. STAT5 and STAT6 are the members of the STAT gene family which have the highest amino acid homology. STAT5 was discovered as a prolactin (PRL) -induced member of the STAT proteins, which is responsible for the PRL-mediated induction of the milk protein genes. The induction of STAT5 by many other cytokines suggested that STAT5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. STAT6 is activated in response to Interleukin-4 (IL-4) and IL-13 and it mediates many transcriptional responses of these cytokines. Cell type- and promoter-specific functions of STATs are most likely modulated by the interaction with other transcription factors and/or coactivators. We investigated the mechanism by which STAT5 and STAT6 induce transcription. Previous studies have shown that diverse regulated transcription factors interact with coactivators such as p300 and CBP. We report that p300/CBP act as coactivators of both STAT5 and STAT6. Their ectopic expression enhances PRL-induced STAT5-mediated transcriptional activation and IL-4 induced STAT6 activated transcription. Consistent with this observation, we found that the adenovirus E1A protein, which binds to p300/CBP, suppresses STAT5- as well as STAT6-induced transcriptional activation.Coimmuno-precipitation and mammalian two-hybrid assays demonstrate that STAT5 and STAT6 directly contact different interaction domains of p300/CBP. STAT5 contacts the CREB binding domain of p300/CBP whereas STAT6 interacts with the C-terminal interaction domain of p300/CBP. These results indicate that, although several STAT family members contact the same coactivator, the mechanism of transcription activation may be different. p300/CBP proteins are complexed with other coactivator proteins and it is postulated that different p300/CBP containing complexes exist. We are currently investigating which subcelluar complex of p300/CBP and which p300/CBP-associated factors are involved in transcriptional regulation by STAT5 and STAT6. DNA methylation in the control of T-cell-specific gene expression:
methylation patterns differ between T cells, other lymphoid and non-
lymphoid cells
Rothenburg, S., Koch-Nolte, F., Thiele, H.-G., Haag, F. Universität Hamburg, Institut für Immunologie, Martinistr. 52, D-20246 Hamburg, Germany Phone: ++49-40-42803 4595, Fax: ++49-40-42803 4243, eMail: haag@uke.uni-hamburg.de Expression of the rat T cell differentiation marker RT6 is restricted to lymphocytes. Within this lineage only mature peripheral T cells, intraepithelial lymphocytes and a subpopulation of natural killer cells express this protein. RT6 is not expressed in thymocytes or recent thymic migrants. Interestingly, in RT6 heterozygous rats, a subpopulation of T cells expresses only one RT6 allele. We have previously shown that this monoallelic expression is determined by cis-regulatory elements. We have now investigated the role of DNA methylation in the regulation of RT6 expression. We found that the RT6 promoter was hypomethylated in RT6 expressing tissues, and methylated in non-expressing ones. Remarkably, among RT6 non-expressing tissues, the extent of methylated regions differed substantially between lymphoid cells, where regions larger than 23 kb were methylated, and non-lymphoid tissues, where methylation was restricted to a 3 to 4 kb region surrounding the promoter. We also found that in cells expressing only one RT6 allele, the two alleles differ in their methylation status. The promoter region of the silent allele was methylated, while that of the transcribed allele was not. In RT6 non-expressing thymoma cells RT6 expression was induced by treatment with the methyltransferase inhibitor 5-azacytidine. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT6 promoter activity, while that of the SV40 promoter was not influenced. Our findings indicate that DNA methylation plays an important role in the allele- and tissue-specific control of RT6 expression, and that it is regulated distinctly in lymphoid and non-lymphoid cells. Evidence for a dual role of p38 kinase: proliferation
vs. cell cycle arrest
Dietrich, C., Faust, D., Schmitt, C., Bongartz, S., and Oesch., F. Institute of Toxicology, Obere Zahlbacherstr. 67, 55131 Mainz, Germany Phone: 06131 / 393 3066, Fax: 06131 / 230506, eMail: cdietric@mail.uni-mainz.de It is generally accepted that p38 kinase which belongs to the family of MAPKs is strongly activated by cellular, e.g. metabolic, stress which finally leads to cell cycle arrest [Science (1996), 272, 1347]. In contrast, we have observed that proliferation of human (FH109) and murine (NIH3T3) fibroblasts can be inhibited by the p38-specific inhibitor SB203580 suggesting a mitogenic role of p38 kinase. We hypothesized that p38 might have a dual role in cell cycle control depending on the kinetics of activation. The aim of our work was therefore to elucidate the mechanisms underlying the different cellular responses after anisomycin- and serum-induced p38 activation in FH109 and NIH3T3 fibroblasts. In both cell lines, anisomycin-treatment (10 µg/ml) induced a strong and sustained activation of p38 while serum-stimulation resulted in a weaker and transient activation of p38 as assessed by Western blotting with a phospho-specific antibody and kinase assays after immunoprecipitation. In agreement with our hypothesis, overexpression of p38 mimicking sustained activation led to a significant decrease in BrdU-incorporation in NIH3T3 cells compared to mock-transfected cells. Immunofluorescence studies revealed a strong and persisting nuclear staining of phosphorylated p38 after anisomycin-treatment. In contrast, phosphorylated p38 was detected in focal contacts, in colocalization with the actin cytoskeleton as well as weakly in the nucleus after serum-stimulation. Phosphorylation of the transription factor CREB was fully inhibited by SB203580 (10 µM) after anisomycin- but not at all after serum-stimulation suggesting that p38 exclusively mediates CREB-phosphorylation induced by metabolic stress. We could further show that p38 is differentially activated by the two stimuli. While anisomycin-induced phosphorylation of p38 is known to be independent of rho-proteins, serum-induced p38 activation is mediated by a rac-dependent pathway as detected by using bacterial toxins. This work was supported by the grant DI 793/1-1 from DFG Signal transduction mechanism leading to the growth inhibition of human
glioblastoma cells by Oncostatin M
Halfter, H., Friedrich, M., Ringelstein, E.B., Stögbauer, F. University of Münster, Neurology, Albert-Schweitzer-Str. 33, 48129 Münster, Germany Phone: 0251-834 8184, Fax: 0251-834 8181, eMail: Halfter@uni-muenster.de Oncostatin M (OSM) is a member of the Interleukin-6 (IL-6) family of cytokines that provokes pleiotropic effects on different cell types. We have recently shown that OSM exerts a potent growth inhibitory effect on several human glioma cell lines. The OSM treated glioma cells aquire a cell morphology characterized by multiple long processes indicating differentiation of the tumor cells into an astrocytic phenotype. We have demonstrated that the OSM treatment of the glioma cells leads to a strong induction of several components of the Jak-Stat pathway as well as the ras-raf-MAPK pathway. In order to investigate the signaling mechanism leading to glioma cell differentiation, we had blocked the Jak-Stat pathway by overexpressing dominant-negative (dn) Stat3. Contrarily to other cells where the effects of IL-6 cytokines were inhibited by dnStat3, differentiation of the glioblastoma cells was not affected by dnStat3. Trying to characterize signal transduction molecules which are essential for the OSM effect in the glioblastoma cells, we expressed dominant-negative proteins of several important signal transducing molecules including dn c-src, dn c-raf1 and dn Shp-2 and examined their effects on OSM-mediated growth inhibition. Furthermore, we tried to reveal the mechanism underlying the cell cycle arrest caused by OSM treatment. In accordance with other investigations, we found an enhanced expression of p27 leads to cell cycle arrest in the G1 phase. Comparing the signal transducing pathways of OSM in several different glioblastoma cell lines, we found that all cell lines involved in this study showed a strong activation of the Jak-Stat pathway. However, only some of these were growth inhibited by OSM, and these cell lines showed an enhanced p27 expression. This led to the conclusion that activation of the Jak-Stat pathway is not sufficient (and probably not necessary) for the OSM-induced expression of p27 and finally to cause growth inhibition of glioma cells. Advanced Applications in Fluorescence Microscopy
Visitron Systems GmbH, Gutenbergstr. 9, 82178 Puchheim, D Phone: ++89-8902450, Fax: ++89-89024518, eMail: gdraude@visitron.de Fluorescence microscopy is one of the most important methods for the observation and analysis of signal transduction and other physiological reactions. As in the beginning, the observation of the reactions was the main issue, today many technics give reproduceable results for the quantitative analysis of the seen fluorescence. Dyes, excitation sources and detection tools become more sophisticated in the last years, so that in-vitro applications can be performed. To localize different structures of interest in the same organism, tissue or single cell, it becomes necassary to investigate into a second (or third) marker. For a good seperation between the structures of a specimen, it is important that the fluorochromes involved does not interfere. Not only the choice of the dye-system for double-labeling, but also the conditions of the optical system are important for a good reproduceable colocalization. Here, the points of major interests are the chararcteristics of the filters and the sensitivity and dynamic of the camera-system used. One of the most common misunderstanding for analysis and documenation of multi-channel flourescence microscopy is the need of a color camera-system. As the emitted wavelength of a fluorochrome is already seperated by the optical filter system of the microscope, a second filter system in front of the the camera-CCD becomes obsolete. In contrast, this reduces the sensitivity of the camera, and additionally the resolution of the single color using a mosaic filter CCD (which in fact is the most used color CCD). However, using dye-specific filter-systems and taking monochrome pictures, the best results will be achieved. To perform colocalization, a powerful imaging-software is needed. Intensity analysis with a spread-sheet output, overlay of brightfield images with several fluorescence channels, and a powerful filter algorithm for deblurring fluorescence background is state of the art. Since 1994 , different mutants of the green fluorescent protein (GFP) improved fluorescence microscopy among molecular, developmental and cell biologists. GFP is a small protein (27 Kd) and the DNA sequences coding for GFP can be manipulated by recombinant DNA technology to create gene fusions between GFP and gene-sequences of any protein of interest. Today, we have several different types of the GFP-family for a multi-channel fluorescence imaging. GFP-dyes improves the fluorescence resonance energy transfer (FRET) technic, used for the detection of intracellular protein-interactions that was beyond the resolution of light-microscopy. In the recent years, FRET microscopy gets a powerful tool to detect the kinetics of physiological reactions due to online-imaging and quantification of the intensity shift of the emission wavelengths. Subsequently, time-depend reactions can also performed with other labeling systems, e.g. Fura and BCECF for Ca2+ and pH, respectively. Therefore, a fast and sensitive imaging-system is needed, which controlls and synchronizes all components of wavelength changes in excitation and emission with the detection system. In conclusion, fluorescence imaging becomes one of the most powerful tool for analysis and observation of cellular reactions due to advanced CCD-camera technics, modern computersystems and software solutions. Workshop E
Cell Cycle and Control of Transcription (Part 2)
Michael Naumann, Berlin
Edith Pfitzner, Frankfurt
Suppressor of cytokine signaling (SOCS)-3 is induced by CpG-DNA in
murine macrophages and suppresses IFN-gamma signal transduction
Dalpke, A., Opper, S., Zimmermann, S., Heeg, K. Inst. for Med. Microbiology, , Pilgrimstein 2, 35037 Marburg, Germany Phone: 06421-2866497, Fax: 06421-2866420 Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine- inducible negative regulators of JAK/STAT signaling in cells of hematopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen derived stimuli we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines with CpG-DNA. Here we show that CpG-DNA but not GpC-DNA induces expression of SOCS-1 and SOCS-3 mRNA and protein. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the MAPK pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA indicating that SOCS expression can be induced by multiple signal pathways distinct from the JAK/STAT pathway. SOCS proteins induced by CpG-DNA were functional since they inhibited IFN-gamma induced phosphorylation of STAT-1 protein as well as IL-6 induced STAT-3 phosphorylation. Hence the results indicate a substantial crosstalk between signal pathways within the cells. They provide evidence for regulative mechanisms of JAK/STAT-signaling after triggering Toll like receptor (TLR)-related signal pathways. ERK5/BMK-1 regulates muscle cell differentiation
Dinev, D., Neufeld, B., Jordan, B.W.M., Lee, J-D., Lindemann, D., Rapp, U.R., Medizinische Strahlenkunde und Zellforschung, Virale Pathologie, Versbacher Str. 5, Phone: 0931/ 2013851, eMail: dragomir.dinev@mail.uni-wuerzburg.de MAP kinase cascades regulate a variety of cellular processes in response to extracellular stimuli, such as proliferation, differentiation, cell activation and apoptosis. Recently a novel member of the MAP kinase family, ERK5/BMK-1 (big MAP kinase), has been identified and shown to be selectively activated by the dual-specificity kinase MEK5. Little is known about the physiological role of this novel MAPK cascade. The fact that ERK5 can phosphorylate members of the MEF2 family of transcription factors, which play an important role in myogenesis, prompted us to examine a possible function of the MEK5/ERK5 cascade during muscle differentiation. As a model system we have used the mouse myoblast cell line C2C12 which is committed to differentiate upon stimulation with 2% horse serum. Under these conditions we have observed a rapid activation of endogenous ERK5. Selective activation of the pathway by overexpression of active MEK5 and ERK5 resulted in upregulation of the p21 promoter, whereas promoter activity remained silent if wildtype ERK5 was replaced by a dominant negative form of the kinase. In addition the MEK5/ ERK5 cascade acts synergistically with MyoD in the transactivation of the myosin light chain-1 promoter and a promoter construct carrying 4 copies of an E-box-element, which is found in the promoters of muscle specific genes. To further analyse the role of ERK5 in myogenesis, C2C12 cells were infected with different retroviruses resulting in the stable transduction and expression of ERK5wt, dominant negative ERK5 or an ERK5-antisense RNA. After induction with horse serum, expression of differentiation markers such as p21, MyoD or myogenin were enhanced in ERK5 overexpressing cells. In contrast, marker expression as well as morphological differentiation was completely abolished in cells which did not allow ERK5 to be expressed due to the presence of an antisense RNA. Taken together our study shows that the MEK5/ ERK5 MAP kinase cascade plays a pivotal role in the early steps of muscle cell differentiation. Apoptosis suppression by Raf-1 and MEK1 requires MEK- and PI-3-
Kinase dependent signals
Alexander von Gise§, Petra Lorenz§, Brian Hemmings#, Ulf R. Rapp§, and Jakob Troppmair§ § Institut für Medizinische Strahlenkunde und Zellforschung (MSZ) # Friedrich Miescher Institute, Basel, Switzerland. Phone: 0931-2013841, Fax: 0931-201383, troppmair@mail.uni-wuerzburg.de Two Ras effector pathways involve the activation of Raf-1 and PI-3-kinase (PI3K) and have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of
apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation
of the kinase in this process. Mitochondrially active Raf-1 is unable to activate ERK1,2 but
suppresses cell death by inactivating the pro- apoptotic Bcl-2 family member BAD. However,
genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-
ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling
using the IL-3 dependent cell line 32D. MEK is essential for survival and growth in the
presence of IL-3. Upon growth factor withdrawal expression of constitutively active MEK1
mutants significantly delays the onset of apoptosis. This protective effect is achieved by both
MEK- and PI3K- dependent mechanisms and results in the phosphorylation of AKT. The
same MEK- and PI3K- dependence is also observed in 32D cells protected from apoptosis by
oncogenic Raf-1. Requirement for the PI3K effector AKT in this process is further
demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1 induced
cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in
apoptosis suppression. In summary these data suggest a Raf effector pathway for cell survival
that is mediated by MEK and AKT.

Pontin52 and Reptin52 antagonistically regulate
-catenin signalling
activity
Huber, O., Tauber, R., Kemler, R. Bauer, A. University Hospital Benjamin Franklin, Dept. of Clinical Chemistry and Pathobiochemistry, Hindenburgdamm 30, 12200 Berlin, Germany Phone: 030-8445-2525, Fax: 030-8445-4152, eMail: Huber@ukbf.fu-berlin.de In cells stimulated by a Wnt-signal,
-catenin becomes stabilized in the cytoplasm and enters the nucleus where it interacts with HMG box transcription factors of the LEF-1/TCF family (1) thereby stimulating the transcription of specific target genes. We recently identified the nuclear protein Pontin52 as an interaction partner of
-catenin suggesting that Pontin52 is involved in the regulation of
-catenin mediated transcriptional activation (2). In a two-hybrid screen we now identified Reptin52 as an interaction partner of Pontin52, that is highly homologous to Pontin52 itself. Reptin52, like Pontin52, is able to self-interact and as previously demonstrated for Pontin52 also interacts with
-catenin and the TATA box binding protein (TPB). To analyze whether Pontin52 and Reptin52 are involved in the regulation of Wnt-target genes, reporter gene assays were performed. Indeed, Pontin52 and Reptin52 modulate the transactivation potential of the TCF/
-catenin complex. Interestingly the two proteins antagonistically regulated gene transcription with Pontin52 having an activating and Reptin52 a repressing function. This can be correlated with the opposing polarity of the ATP-dependent helicase activity of both proteins (3). Moreover we show that this regulatory function depends on the interaction of Pontin52 and Reptin52 with
-catenin since the artificial activator LEFN-VP16 that does not require
-catenin for transactivation was not influenced. Thus Pontin52 and Peptin52 represent important modulatory proteins that are involved in regulation of Wnt-target gen transcription. Recent reports indicate that Pontin52 and Reptin52 are involved in chromatin remodelling processes (4) that also appeared to be essential for c-Myc oncogenic acitvity (5). (1) Huber et al., 1996, Mech. Dev. 59, 3-10. (2) Bauer et al. , 1998, Proc. Natl. Acad. Sci. USA 95, 14787-14792. (3) Kanemaki et al., 1999, J. Biol. Chem. 274, 22437-22444. (4) Wood et al., 2000, Mol. Cell, 5, 321-330. (5) Shen et al., 2000, Nature, 406, 541-544. GABP/
complex as the regulator of IL-2receptor 
 chain expression
Avots, A., Reimer, O., Stibbe, C., Schulze, J.,. and Serfling, E. Universität Würzburg, Molekulare Pathologie, J-Schneider Str. 2, D-97080 Würzburg Phone: 0931-201-3495, Fax: 0931-201-3429, eMail: path061@mail.uni-wuerzburg.de Interleukin-2 (IL-2) and IL-2 receptors (IL-2R) regulate the magnitude, duration and termination of the T cell response following antigen stimulation. Different combinations of the IL2R ,
and  chains result in the formation of three different classes of IL-2Rs. IL-2R-chain is not expressed on resting T-cells and IL-2R
-chain is expressed at low levels. Upon the stimulation expression levels of  and
chains are drastically up-regulated. IL-2R-chain, also known as the 'common -chain', is constitutively expressed. While several inducible transcription factors (e.a. NF-ATs, STAT5, GATA, and NF-B) were shown to control the activation of - and
-chain genes, GABP
complex with the constitutive DNA-binding activity was postulated as a positive regulator of
- and -chain gene expression. In order to investigate the regulation of IL-2R,
- and -chain expression at the level of intact chromosomal genes we have created retrovirally transduced murine El-4 T-cell line over-expressing GABP. RNase protection assays revealed slightly up-regulated -chain levels and completely abolished inducible expression of - and
-chains. As GABP subunit does not contain a transcription activation domain, these results would imply that GABP
complex differentially regulate
- and -chain expression. Unexpected down-regulation of -chain expression prompted us to re-investigate the regulation of the promoter and IL-2-responsive upstream element (IL-2RE) of IL-2R-chain gene. The activity of IL-2RE depends on three critical regions, composed of STAT, GATA and Ets-motifs. Our DNA-binding experiments show strong competition between GABP
and Elf-1, previously identified as the true regulator of the Ets-motif within the IL-2RE. Taken together our results suggest the critical involvement of GABP
complex in the regulation of all IL-2R chains. Ongoing retroviral infections of murine primary cells will enable us to elucidate the effect of GABP
overexpression and functional deficiency on the activation, proliferation, differentiation and apoptosis of T-cells.

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