Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase. Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase. leukemia inhibitory factor epidermal growth factor extracellular signal regulated kinase c-Jun N-terminal kinase mitogen-activated protein kinase mitogen-activated protein kinase-activated protein mitogen stress kinase protein kinase C ribosomal S6 kinase MAP/ERK kinase cAMP-responsive element-binding protein signal transducer and activator of transcription embryonic stem fetal calf serum c-Sis-induced element wild type Leukemia inhibitory factor (LIF)1 is a pleiotropic cytokine that belongs to the interleukin 6 cytokine family including ciliary neurotrophic factor, oncostatin M, and cardiotrophin-1. It is secreted by various cell types and mediates opposite effects (either proliferative or differentiative) depending on the cell lineages and stage of differentiation (1Hilton D.J. Trends Biochem. Sci. 1992; 17: 72-76Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 2Shellard J. Perreau J. Brulet P. Eur. Cytokine Netw. 1996; 7: 699-712PubMed Google Scholar, 3Taupin J.L. Pitard V. Dechanet J. Miossec V. Gualde N. Moreau J.F. Int. Rev. Immunol. 1998; 16: 397-426Crossref PubMed Scopus (62) Google Scholar). LIF also influences the survival, differentiation, and response to injury of neuronal cell lineages and synergizes with ciliary neurotrophic factor for motoneuron cell survival (4Aloisi F. Rosa S. Testa U. Bonsi P. Russo G. Peschle C. Levi G. J. Immunol. 1994; 152: 5022-5031PubMed Google Scholar, 5Sendtner M. Gotz R. Holtmann B. Escary J.L. Masu Y. Carroll P. Wolf E. Brem G. Brulet P. Thoenen H. Curr. 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Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar, 10Hirano T. Nakajima K. Hibi M. Cytokine Growth. Factor. Rev. 1997; 8: 241-252Crossref PubMed Scopus (327) Google Scholar, 11Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1227) Google Scholar, 12Ernst M. Novak U. Nicholson S.E. Layton J.E. Dunn A.R. J. Biol. Chem. 1999; 274: 9729-9737Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However the identity of LIF-induced proteins in different cell contexts has not been precisely characterized. One major goal in this field is to identify LIF-dependent pathways in LIF-sensitive cell lines derived from the same founder cells in which LIF may trigger various effects. The most appropriate cells to study LIF signaling (which may satisfy these criteria) are the mouse embryonic stem (ES) cells. These cells are derived from the inner cell mass of blastocysts, and they remain pluripotent in vitro when maintained in the presence of LIF (13Brook F.A. Gardner R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5709-5712Crossref PubMed Scopus (439) Google Scholar, 14Bradley A. Zheng B. Liu P. Int. J. Dev. Biol. 1998; 42: 943-950PubMed Google Scholar). Upon LIF withdrawal, ES cells differentiate heterogeneously into various cell types, and some of the cells die by apoptosis during the differentiation process (15Duval D. Reinhardt B. Kedinger C. Boeuf H. FASEB J. 2000; 14: 1577-1584Crossref PubMed Google Scholar). ES cells can also be induced to differentiate homogeneously into determined cell types (16Dinsmore J. Ratliff J. Deacon T. Pakzaban P. Jacoby D. Galpern W. Isacson O. Cell Transplant. 1996; 5: 131-143Crossref PubMed Scopus (166) Google Scholar, 17Li M. Pevny L. Lovell-Badge R. Smith A. Curr. 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A. 1998; 95: 11107-11112Crossref PubMed Scopus (201) Google Scholar, 52Woetmann A. Nielsen M. Christensen S.T. Brockdorff J. Kaltoft K. Engel A.M. Skov S. Brender C. Geisler C. Svejgaard A. Rygaard J. Leick V. Odum N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10620-10625Crossref PubMed Scopus (119) Google Scholar, 53Jain N. Zhang T. Kee W.H. Li W. Cao X. J. Biol. Chem. 1999; 274: 24392-24400Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). To get insight into LIF signaling in mouse ES cells, we have followed the LIF-dependent phosphorylation profiles and DNA binding activities of the STAT3 transcription factor under different ES cell growth conditions: "pluripotent," in the continuous presence of LIF; "reversibly differentiation-committed," without LIF for 20 h, and "irreversibly differentiation-committed," 48 h without LIF. We have also characterized the activation profile of MAPKs and identified new LIF targets (RSKs and CREB) regulated by ERKs, whose role in the maintenance of ES cell pluripotency is discussed under "Discussion." Also, by using Ser/Thr kinase inhibitors, we have distinguished LIF-induced-ERK and PKC-dependent pathways. In addition, activation profiles of some of these LIF-induced proteins have also been studied in an ES-derived neuronal cell line that we have characterized as being a LIF-sensitive cell line. ES cells were derived from the inner cell mass of mouse blastocysts as described (54Hogan B.L. Beddington R. Costantini F. Lacy E. A Laboratory Manual. Second Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 255-272Google Scholar). The ES S1 cell line, grown in LIF-containing medium without feeder cells, was used in these experiments unless indicated. The ES H1 wild type (WT) and the derived ES H1RSK2− (X/Y cells in which the X-linkedrsk2 gene has been deleted by homologous recombination) were grown on feeder cells, in the presence of LIF (55Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (423) Google Scholar). In this RSK2− cell line, shortened mRNA corresponding to the in-frame skipping of exon 2 (in which the neomycine resistance gene and the stop codons were inserted) has been detected by reverse transcriptase-polymerase chain reaction analysis, indicating that the RSK2− cell line used in this study may express a hypomorph allele of rsk2. However, no RSK2 protein was detected in this cell line under classical Western blot conditions. H1-derived cell lines were passaged twice without feeder cells in the continuous presence of LIF prior to LIF withdrawal and reinduction. The polyclonal anti-phospho Ser-727-STAT3 (56Frank D.A. Mahajan S. Ritz J. J. Clin. Invest. 1997; 100: 3140-3148Crossref PubMed Scopus (216) Google Scholar), anti-phospho Tyr-705-STAT3 (QCB/BioSource International), anti-STAT3, ERK2 (C-14), ERK1 (C-16), and RSK2 (E-1) (Santa Cruz Biotechnology); the anti-phospho-JNK1/JNK2 and anti-phospho-p38 (Cell Signaling); the monoclonal anti-phospho-ERK1/ERK2 (Biolabs) and anti-JNK1 (PharMingen) antibodies; and the U0126 (Promega) and H7 (Biomol) compound were used as recommended by the manufacturers. The monoclonal anti-phospho Thr-577-RSK antibody has been described (29Merienne K. Jacquot S. Zeniou M. Pannetier S. Sassone-Corsi P. Hanauer A. Oncogene. 2000; 19: 4221-4229Crossref PubMed Scopus (38) Google Scholar). When indicated, quantification of the signals has been performed with the Bio-Rad GS700 imaging densitometer by using Molecular Analyst, version 2.1, software. ES cells were grown in medium without LIF (10 ml/10-cm diameter Petri dish of Dulbecco's modified Eagle's medium, 4,5 g/l glucose, 10% FCS, 862 mg/ml glutamax, 0.1 mm β2-mercaptoethanol) for various time periods extending from 6 to 72 h and reinduced with LIF (1000 units/ml) up to cell lysis, which was performed 72 h after the beginning of the experiment. Apoptosis is scored by the appearance of a DNA ladder following DNA extraction by the Hirt procedure as described previously (15Duval D. Reinhardt B. Kedinger C. Boeuf H. FASEB J. 2000; 14: 1577-1584Crossref PubMed Google Scholar). S1 cells were grown in the continuous presence of LIF, and medium was changed every other day. Cell lysates were prepared without medium change or 15 min after LIF-containing medium addition (pluripotent conditions). ES cells grown without LIF for 20 h (differentiation-committed, reversible) or 48 h (differentiation-committed, irreversible) were re-fed with LIF-containing medium 15 min before harvesting. Cell lysates were prepared from cells grown under these different conditions. Neuronal differentiation of ES cells was mainly induced as described (16Dinsmore J. Ratliff J. Deacon T. Pakzaban P. Jacoby D. Galpern W. Isacson O. Cell Transplant. 1996; 5: 131-143Crossref PubMed Scopus (166) Google Scholar) with minor modifications. The S1 ES cell line was grown as embryoid bodies (300,000 cells/ml) on bacterial Petri dishes in Dulbecco's modified minimal essential medium (Life Technologies, Inc.) supplemented with 15% fetal calf serum (Hyclone), 0.1 mm β2 mercaptoethanol, 0.1% non-essential amino acids (Life Technologies, Inc.), 50 units/ml penicillin, and 50 μg/ml streptomycin (Life Technologies, Inc.) in the presence of 10−6mall-trans-retinoic acid (Sigma). After 4 days of retinoic acid treatment, the embryoid bodies were trypsinized, and the cells were resuspended in the same medium. 40,000 cells/ml were then plated in Dulbecco's modified Eagle's medium on coated culture dishes (for biochemical studies) or glass coverslips (for immunocytochemistry). Coating was performed successively with 0.1% gelatin, 10 μg/ml poly-d-lysine, and 1 μg/ml laminine. After 2 h, the culture medium was replaced by a defined medium (16Dinsmore J. Ratliff J. Deacon T. Pakzaban P. Jacoby D. Galpern W. Isacson O. Cell Transplant. 1996; 5: 131-143Crossref PubMed Scopus (166) Google Scholar). The cell medium was changed every other day. LIF induction (30 min with 250 or 500 units of LIF/ml) was performed on homogeneous neuronal cell populations 4 days after plating. Cells (plated on glass-coated coverslips, coated as described above) fixed with 4% paraformaldehyde (15 min) were treated with 3% H2O2 and permeabilized with 0.1% Triton X-100 (10 min). After preincubation with 10% normal goat serum for 30 min, cells were treated with rabbit antiserum directed against Neurofilament 200 (Sigma) diluted 1:100 or mouse antiserum directed against MAP1 (Sigma) diluted 1:300. Peroxidase-conjugated anti-rabbit and anti-mouse antibodies were used as secondary antibody, respectively. ES cells grown without LIF for 20 h were pretreated for 1 h with 50 μm H7 or various concentrations of U0126, before reinduction with LIF in the presence of the inhibitors, at the same concentrations. Cytosolic and nuclear cell lysates were prepared as described (57Sadowski H.B. Gilman M.Z. Nature. 1993; 362: 79-83Crossref PubMed Scopus (234) Google Scholar). Whole cell lysates were a mixture of cytosolic and nuclear lysates at a 2:1 ratio. Band shift experiments were performed with the nuclear lysates (30 μg) on the high affinity STAT3 DNA binding site of the c-fos promoter (c-Sis-induced element (SIE)) as described (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar). Nuclear or whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes in the presence of 0.07% SDS. Proteins were reacted with the different antibodies as recommended by the manufacturers. Mouse ES cells were grown in the continuous presence of LIF and were fed with fresh medium or passaged every other day. In the absence of LIF, ES cells are committed to differentiation. Analysis of the expression profile of genes know as pluripotent (rex-1,fgf4, esp) or "differentiated" cell markers (fgf-5) indicates that by 24 h of LIF withdrawal, cells are committed to differentiate (58Scherer C.A. Chen J. Nachabeh A. Hopkins N. Ruley H.E. Cell Growth Differ. 1996; 7: 1393-1401PubMed Google Scholar, 59Ben-Shushan E. Thompson J.R. Gudas L.J. Bergman Y. Mol. Cell. Biol. 1998; 18: 1866-1878Crossref PubMed Scopus (215) Google Scholar, 60Lake J. Rathjen J. Remiszewski J. Rathjen P.D. J. Cell Sci. 2000; 113: 555-566Crossref PubMed Google Scholar). 2D. Duval, B. Reinhardt, C. Kedinger, and H. Boeuf, submitted for publication. In addition, ES cells grown without LIF for 48 h have lost their pluripotency, as suggested by their inability to colonize embryos (61Burdon T. Stracey C. Chambers I. Nichols J. Smith A. Dev. Biol. 1999; 210: 30-43Crossref PubMed Scopus (463) Google Scholar). We have developed a quicker test to determine the point of irreversible commitment of cell differentiation when cells are grown in the absence of LIF. Based on our previous observation that during the differentiation process 30% of the cells are dying by apoptosis (15Duval D. Reinhardt B. Kedinger C. Boeuf H. FASEB J. 2000; 14: 1577-1584Crossref PubMed Google Scholar),2 we have set up an in vitro pluripotency test: cells were grown in the absence of LIF for different time periods (between 6 and 72 h) and then re-fed with LIF-containing medium up to 72 h before harvesting. Cells were scored for apoptosis by the appearance of a DNA ladder, a qualitative test that allows rapid detection of apoptotic cells (Fig. 1). The morphology of the cells and integrity of their DNA were unaltered when LIF was withdrawn for 24 h, compared with cells maintained in the continuous presence of LIF. By contrast, from 36 h onward after LIF withdrawal, ES cell clumps start to dissociate and cells begin to spread, reflecting the differentiation process. Meanwhile, dying cells were detected, and increasing proportions of DNA were degraded, indicating that some of the cells were dying by apoptosis. Therefore, we conclude that ES cells are irreversibly committed to differentiation and/or apoptosis starting at 36 h after LIF withdrawal. Based on this in vitro test, as well as on in vivo data (14Bradley A. Zheng B. Liu P. Int. J. Dev. Biol. 1998; 42: 943-950PubMed Google Scholar, 61Burdon T. Stracey C. Chambers I. Nichols J. Smith A. Dev. Biol. 1999; 210: 30-43Crossref PubMed Scopus (463) Google Scholar), we will refer throughout the document to pluripotent, reversibly differentiation-committed, or irreversibly differentiation-committed cells. STAT3 is critical for the maintenance of pluripotent ES cells as well as in various LIF-sensitive differentiated cells (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar,62Megeney L.A. Perry R.L.S. Lecouter J.E. Rudnicki M.A. Dev. Genet. 1996; 19: 139-145Crossref PubMed Scopus (80) Google Scholar, 63Bonni A. Sun Y. Nadal-Vicens M. Bhatt A. Frank D.A. Rozovsky I. Stahl N. Yancopoulos G.D. Greenberg M.E. Science. 1997; 278: 477-483Crossref PubMed Scopus (847) Google Scholar, 64Kunisada K. Tone E. Fujio Y. Matsui H. Yamauchi-Takihara K. Kishimoto T. Circulation. 1998; 98: 346-352Crossref PubMed Scopus (201) Google Scholar). Phosphorylation of STAT3 at the Tyr-705 residue is crucial for its activity in ES cells (11Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1227) Google Scholar, 38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar). It is also phosphorylated at the Ser-727 MAPK consensus site in response to various stimuli such as EGF or interleukin 6. We were interested to follow the STAT3 activation profile under experimental conditions as defined above. In these experiments, LIF was withdrawn for 20 or 48 h as a way to determine LIF-dependent STAT3 activation at a very early stage of differentiation commitment, whereas the process is still reversible (20 h after LIF withdrawal) as well as in irreversibly differentiation-committed cells (48 h after LIF withdrawal). Western blot analyses of nuclear extracts from ES cells grown under these conditions were performed using specific antibodies that recognize the activated STAT3 proteins phosphorylated on the Tyr-705 (P-Y705-STAT3) or Ser-727 (P-S727-STAT3) residues (Fig. 2). Phosphorylated STAT3 is detected in the continuous presence of LIF with a clear enhancement of the level of phosphorylation when fresh medium with LIF is added (Fig.2, Pluripotent cell growth conditions). By contrast, no phosphorylation on Tyr-705 and Ser-727 residues is detected in ES cells grown in the absence of LIF for 20 or 48 h. However, a rapid phosphorylation at both sites is induced upon LIF addition, indicating that these cells are still LIF-responsive (Fig. 2, Dif. com. at 20 h and Dif. com. at 48 h cell growth conditions). STAT3 phosphorylation is correlated with its specific DNA binding activity, as detected on the SIE probe in the Pluripotentand Dif. com. cells (Fig. 2). Protein-DNA complex formation on a LIF-unresponsive site was constitutive and unchanged in both cell types, indicating that cell extracts were equally functional (Ref. 38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholarand data not shown). However, we noticed a reduction in the amount of STAT3-dependent DNA binding complexes formed in Dif. com. cells at 48 h upon LIF induction. Also, we have found that the cytosolic extracts (in which phosphorylated STAT3 could be detected (44Stephens J.M. Lumpkin S.J. Fishman J.B. J. Biol. Chem. 1998; 273: 31408-31416Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar)) did not exhibit specific DNA binding activity, indicating that nuclear partners may stabilize the DNA-phospho-STAT3 complexes (data not shown). These results indicate that LIF-induced STAT3-dependent complexes, which include phosphorylated STAT3 proteins, are present in ES cells grown under the pluripotent and differentiation-committed cells. We have analyzed the activation profile of members of the three MAPK families (ERK, JNK, and p38) in ES cells. Western blot analysis of total cell lysates from ES cells maintained under various conditions was performed with antibodies against MAPKs that specifically recognize the activated dually phosphorylated forms of ERK1/ERK2 (P-ERK1/P-ERK2, P-ERKs), JNK1/JNK2 (P-JNK1/P-JNK2), and p38 (P-p38). As shown in Fig.3 A, the ERK1 and ERK2 proteins are induced by LIF in cells maintained in the presence of low or high serum concentrations, which is in good agreement with previous studies (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar, 49Ernst M. Oates A. Dunn A.R. J. Biol. Chem. 1996; 271: 30136-30143Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). However, MAPKs (as well as their targets, such as the RSK proteins) are serum-induced proteins (25Frodin M. Gammeltoft S. Mol. Cell. Endocrinol. 1999; 151: 65-77Crossref PubMed Scopus (611) Google Scholar). Therefore, it was of interest to test the direct effect of serum and LIF independently because both are required for the proper growth of ES cells. 3H. Boeuf, D. Duval, B. Reinhardt, and C. Kedinger, unpublished observations. We were also interested in determining the sensitivity of the various LIF-dependent targets in the presence of the U0126 compound, a specific MAP/ERK kinase (MEK) inhibitor that acts downstream of MEK1 and MEK2, impairing phosphorylation of ERK1 and ERK2 at the specific TEY site (65Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2728) Google Scholar, 66Tolwinski N.S. Shapiro P.S. Goueli S. Ahn N.G. J. Biol. Chem. 1999; 274: 6168-6174Abstract Full Text Full Text PDF PubMed Scopu
The adenovirus EIa-inducible early EIIa (EIIaE) promoter is comprised of several sequence elements essential for constitutive and Induced expression. We report here the purification of the host-cell factor that interacts with the major upstream element of this promoter, extending between positions -90 and -70 with respect to the main EIIaE cap site and exhibiting enhancer properties. The puri- fied factor, which corresponds to a 40- to 43-kDa polypeptide, specifically binds to its recognition site and stimulates EIIaE promoter activity when added to an in vitro transcription system, reconstituted from purified factors and RNA polymer- ase. The implication of this factor in the control of the other adenovirus early genes is discussed. Efficient transcription of the adenovirus early transcription units requires the presence of the viral pre-early EIa gene products (1, 2). The mechanism of this transactivation of the early transcription unit is still poorly understood. Extensive deletion and linker scanning mutational analysis (3-5) of the EIa-inducible EIIa early (EIIaE) promoter has indicated that EIa responsiveness involves the same promoter sequences as those required for uninduced expression, suggesting that the same host-cell transcription factors are required in each case. DNA binding studies have identified several of these factors, among which those recognizing sequence elements located between -90 and - 70 (EIIAE-EF (6), EIIaE-B (7,
STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.
ABSTRACT The adenovirus Ela-inducible early ElIa(EIIaE) promoter is comprised of several sequence elementsessential for constitutive and induced expression. Wereportherethe purification ofthe host-cell factor that interacts withthe major upstream element of this promoter, extendingbetween positions -90 and -70 with respect to the mainEIaEcapsite andexhibiting enhancer properties. Thepuri-fied factor, whichcorrespondstoa40-to43-kDapolypeptide,specifically binds to its recognition site and stimulates EIIaEpromoter activity when added to an in vitro transcriptionsystem,reconstitutedfrompurifiedfactorsandRNApolymer- ase. Theimplication of this factor in the control ofthe otheradenovirusearly genes is discussed.Efficient transcription of the adenovirus early transcriptionunits requires the presence of the viral pre-early EIageneproducts(1, 2). Themechanismofthis transactivation oftheearly transcription unit is still poorlyunderstood. Extensivedeletion andlinkerscanningmutational analysis (3-5) oftheEIa-inducibleEllaearly(EIlaE)promoterhasindicatedthat
Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.
The adenovirus EIa-inducible early EIIa (EIIaE) promoter is comprised of several sequence elements essential for constitutive and induced expression. We report here the purification of the host-cell factor that interacts with the major upstream element of this promoter, extending between positions -90 and -70 with respect to the main EIIaE cap site and exhibiting enhancer properties. The purified factor, which corresponds to a 40- to 43-kDa polypeptide, specifically binds to its recognition site and stimulates EIIaE promoter activity when added to an in vitro transcription system, reconstituted from purified factors and RNA polymerase. The implication of this factor in the control of the other adenovirus early genes is discussed.
Journal Article Specific cellular proteins bind to critical promoter sequences of the adenovirus early EIIa promoter Get access Hélène Boeuf, Hélène Boeuf Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Génie Génétique et de Biologie Moléculaire de 1'INSERM, Faculté de Médecine - 11rue Humann - 67085 Strasbourg Cédex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Deborah A. Zajchowski, Deborah A. Zajchowski Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Génie Génétique et de Biologie Moléculaire de 1'INSERM, Faculté de Médecine - 11rue Humann - 67085 Strasbourg Cédex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Taka'aki Tamura, Taka'aki Tamura Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Génie Génétique et de Biologie Moléculaire de 1'INSERM, Faculté de Médecine - 11rue Humann - 67085 Strasbourg Cédex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Charlotte Hauss, Charlotte Hauss Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Génie Génétique et de Biologie Moléculaire de 1'INSERM, Faculté de Médecine - 11rue Humann - 67085 Strasbourg Cédex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Claude Kédinger Claude Kédinger Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Génie Génétique et de Biologie Moléculaire de 1'INSERM, Faculté de Médecine - 11rue Humann - 67085 Strasbourg Cédex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 15, Issue 2, 26 January 1987, Pages 509–527, https://doi.org/10.1093/nar/15.2.509 Published: 26 January 1987 Article history Received: 04 October 1986 Revision received: 15 December 1986 Accepted: 22 December 1986 Published: 26 January 1987
