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T., Yoshida, K., Sudo, T., Naruto, M., and Kishimoto, T. (1994). Molecular cloning of APRF, a novel IFN-stimulated gene factor. 3 p91-related transcription factor ...
Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF) -dependent embryonic stem cell survival DAVID DUVAL, BE´ATRICE REINHARDT, CLAUDE KEDINGER,1 AND HE´LE`NE BOEUF Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (CNRS/INSERM/ULP), BP 163, F-67404 ILLKIRCH Cedex, France ABSTRACT Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF withdrawal results in progressive ES cell differentiation. Here we show that during this differentiation process, part of the cells undergo apoptosis concomitant with an activation of the p38 MAP kinase. To gain insight into events mediated by LIF in ES cells, the expression of potential candidate genes was analyzed in the absence or presence of this cytokine by using a semiquantitative RT-PCR assay. We focused on early response genes and on a new type of cytokine repressors (the Socs proteins), some of which exhibit anti-apoptotic properties. We found that expression of c-Fos, c-Jun, and JunB was induced upon LIF treatment whereas that of JunD, the tyrosine phosphatase ESP, and the components of the LIF receptor remained unaffected. Expression of Socs-3, but not Socs-1 or Socs-2, was stimulated in the presence of LIF. Finally, uncontrolled overexpression of Socs-1 and Socs-3 led to repression of LIF-dependent transcription and severely reduced cell viability, suggesting that the disturbance of a well balanced Socs protein content has adverse effects on cell survival.—Duval, D., Reinhardt, B., Kedinger, C., Boeuf, H. Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF) -dependent embryonic stem cell survival. FASEB J. 14, 1577–1584 (2000)

Key Words: pluripotency 䡠 apoptosis 䡠 early response genes 䡠 transcription

Embryonic stem (ES) cells are pluripotent cells derived from mouse blastocysts that are widely used to study gene functions in muso (1, 2). ES cells can be propagated and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF). This cytokine belongs to a family including interleukin 6 (IL-6), ciliary neurotrophic factor, oncostatin M, and cardiotrophin 1, whose functions are partly redundant and that transduce their signal through the common gp130 receptor subunit in association 0892-6638/00/0014-1577/$02.25 © FASEB

with specific partners (3, 4). Studies performed with knockout mice pointed to the specificity of the different member of this cytokine family of receptors (5– 8). LIF activates at least two pathways: the Janus kinase/signal transducer and activator of transcription (JAK/STAT) and the ras/mitogen activated protein (MAP) kinases pathways, which may converge, leading to phosphorylation/translocation to the nucleus of latent STAT family members and to the transcriptional increase of early response genes such as c-fos, c-jun, junB, and junD (9 –12). Phenotypic analysis of STAT mutant mice and in vitro biochemical studies in different cell systems have emphasized the essential contribution of STAT proteins to cell proliferation and differentiation (13). MAP kinases are serine/threonine kinases activated by growth factors, stress signals, and cytokines and are classified into three families: extracellular regulated kinase (ERK), jun N-terminal kinase (JNK), and p38. They are involved in cell proliferation, differentiation, and apoptotic processes (14 –16). Characterization of the LIF signaling pathways in ES cells is an essential step toward understanding cell pluripotency. Also, comparative studies of gene expression in cell types in which LIF mediates opposite effects (e.g., ES and the myeloid M1 cell lines) should help to characterize the critical components of LIF cell-specific responsiveness. We have previously shown that the ERK2 MAP kinase and the STAT3 transcription factor are activated on LIF treatment in undifferentiated ES cells (17). In addition, it has been demonstrated that STAT3 impairs LIF-dependent differentiation of myeloid M1 cells while it maintains LIF-dependent pluripotency of ES cells (17–20). Furthermore, the early embryonic lethal phenotype of mice lacking STAT3, which die before gastrulation, demonstrates that STAT3 is critical for the early development of mouse embryos (21). 1 Correspondence: IGBMC, 1 rue Laurent Fries, B.P. 163, 67404 ILLKIRCH, France. E-mail: [email protected]

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The recent characterization of the Socs proteins (suppressors of cytokine signaling), which repress LIF signaling, further accounts for the fine-tuning of the pleiotropic effect of LIF. These proteins have been isolated independently as JAK binding proteins, new Src homology-2 (SH2) -containing proteins, or repressors of cytokine signaling (22, 23). They harbor conserved features like a central SH2 domain directly involved in the association with JAK kinases, and a carboxyl-terminal Socs box whose function remains controversial (24, 25). The less conserved amino-terminal part of these Socs proteins may be responsible for their specificity (26 – 29). Forced expression of specific members of the Socs family impaired the LIF-dependent differentiation of the M1 myeloid cell line, probably by an irreversible inactivation of STAT proteins (27, 30). In the present study, we report on a new function of the LIF in ES cells and analyze the expression of selected genes that may be involved in the control of LIF signaling in ES cells. We also investigate the role of particular Socs genes (Socs-1 and Socs-3) in LIF-dependent processes in ES cells.

Transfection and CAT assays ES cells were transfected and CAT assays performed as described (17). Briefly, cells grown in 9 cm Petri dishes were transfected with 5 ␮g of the LIF-responsive (SIE)3-TK CAT reporter vector or 15 ␮g of the (SIEm)3-TK CAT (to clearly visualize basal transcription and its potential repression), together with 1 ␮g of the Socs expression vectors or the empty vector. The percentage of chloramphenicol acetylation was determined from at least three independent experiments, and quantitated with a Bioimaging analyzer (Fuji Photo Film Co.). DNA analysis For DNA extraction (Hirt technique), the cells were directly lysed in ETS buffer (10 mM EDTA, 10 mM Tris-HCl, pH 8, 1% SDS), 4 ml/9 cm petri dish. Cell lysates were supplemented with NaCl (1 M final), left overnight at 4°C, and spun 10 min at 8000 g. The supernatant was treated 3 h with proteinase K (100 ␮g/ml) and the DNA was precipitated with ethanol after phenol/chloroform extraction. After RNase treatment (10 ␮g/ml, Sigma), 30 min at 37°C, 20 ␮g of DNA was loaded on 2% agarose gels and stained with ethidium bromide. Floating cells were recovered by centrifugation and the DNA was prepared as above. Semiquantitative RT-PCR

MATERIALS AND METHODS Cells and lysates The embryonic S1 cell line was derived from the inner cell mass of mouse blastocysts and grown as described (17, 31). Nuclear cell lysates were prepared as described (32). Western blot Nuclear cell lysates were resolved by sodium dodecyl sulfate (SDS)-gel electrophoresis and transferred on nitrocellulose membranes. Proteins were reacted with the anti-PARP (rabbit polyclonal antibody, a gift from G. De Murcia, ESBS, Strasbourg), the anti-phospho-p38 (Promega, Madison, Wis.), or the anti-STAT3 H190X (Santa Cruz Biotechnology, Santa Cruz, Calif.) antibodies. Reporter and expression vectors The (SIE)3-TK-CAT and (SIEm)3-TK-CAT reporter vectors contain the trimerized high-affinity, wild-type (SIE67, sisinduced element) or mutated (SIE25) STAT binding sites inserted upstream of the minimal TK-CAT reporter gene construct (17, 33). The expression vectors S1WT, S3WT, S3⌬N, S3⌬C, and S3⌬NC were constructed as follows. Polymerase chain reaction (PCR) -amplified fragments corresponding to the full-length Socs-1 and Socs-3 cDNAs were cloned into the NdeI/BamHI sites of the p513HA expression vector, a SV40 promoter-based vector in which the sequences inserted were in-frame with the hemagglutinin (HA) tag (a gift from B. Chatton, IGBMC, Strasbourg). The mutants S3⌬N (in which residues 1 to 28 are deleted), S3⌬C (in which residues 176 to 224 are deleted) and S3⌬NC (in which residues 1 to 28 and 176 to 224 are missing) were constructed by PCR amplification of the proper fragments from the S3WT construct. Each construct was verified by sequencing on both DNA strands. 1578

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Total RNA from ES cells was prepared with the Trizol reagent kit (Life Technologies, Inc./BRL), and treated with DNase, (5 U/100 ␮g RNA, Sigma). Total RNA (4 ␮g) was reversetranscribed (RT) with random hexameric primers and the MLV Reverse Transcriptase (Sigma). The RT reaction was split into 4 PCR reactions with 4 sets of specific primers. One PCR reaction was performed with primers corresponding to the 36B4 LIF-independent gene (34) with each RNA preparation. PCR products (20 cycles) were analyzed by Southern blot with random-labeled cDNA fragments. The primers used for the LIF receptor ␤ subunit, gp130 and ESP have been described (35). GenBank accession numbers and primers for the other genes are: junB (U20735): 5⬘primer:ACCTGGCGGATCCCTATCGG 3⬘primer:GCTCCGGACCAGCATAGACG; junD (X15358): 5⬘primer:GAGCAGCATGCTGAAGAAAG 3⬘primer:AGCTGGCTTTGCTTGTGCAG; c-jun (J04115): 5⬘primer:CAGTCTGAAGCCGCACCTCC 3⬘primer:GTTGCTGAGGTTGGCGTAGACC; c-fos (J00370): 5⬘primer:GGCTCTCCTGTCAACACACA 3⬘primer:CCGCTTGGAGTGTATCTGTC; 36B4 (X15267): 5⬘primer:ATGTGAAGTCACTGTGCCAG 3⬘primer:GTGTAATCCGTCTCCACAGA; SOCS1 (U88325): 5⬘primer:GGCAGCCGACAATGCGATCT 3⬘primer:GATCTGGAAGGGGAAGGAAC; SOCS2 (U88327): 5⬘primer:GTTGCCGGAGGAACAGTCCC 3⬘primer:ATGCTGCAGAGTGGGTGCTG; SOCS3 (U88328): 5⬘primer:CGCCTCAAGACCTTCAGCTC 3⬘primer:CTGATCCAGGAACTCCCGAA. Selection of stably transformed cells To establish ES cells that produce the wild-type or mutated Socs proteins, ES cells maintained in LIF-containing medium in 9 cm petri dishes were cotransfected with 10 ␮g of the S1WT, S3WT, and S3⌬NC vectors, together with 4 ␮g of the

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Figure 1. LIF prevents apoptosis in ES cells. A) Constrast phase pictures of ES cells (160⫻) cultivated for several days after LIF withdrawal, as indicated. The arrow points to a dying refringent cell. B) 20 ␮g of DNA were loaded on a 2% agarose gel and stained with ethidium bromide after electrophoresis. DNA was prepared from cells still attached on the dishes (lanes 1– 4, 6, 8, and 9) and from floating cells (lanes 5 and 7). The size of DNA fragments is indicated (M).

PGK-neo selection vector. After 15 days of selection in the presence of 150 ␮g/ml of G418, individual clones were counted. Cotransfection efficiency was determined with an SV40-lac Z plasmid (10 ␮g) transfected together with 4 ␮g of PGK neo (36). After 15 days in the presence of 150 ␮g/ml of G418, stable clones were stained with X-gal. About 65% of the neomycin-resistant clones were positive for ␤-galactosidase expression, indicating that cotransfection efficiency was at least 65%.

RESULTS LIF prevents apoptosis in ES cells The pluripotent feeder-free S1 cell line we used was maintained continuously in the presence of LIF. We have reproducibly observed that withdrawal of LIF from the culture medium drives the cells into a critical phase, during which 30 to 50% of the cells die and that precedes full differentiation of the surviving cells. To examine whether cell death occurred by apoptosis, DNA was prepared from cells at different time periods after LIF withdrawal and analyzed by gel electrophoresis. As shown in Fig. 1, 3 days after LIF withdrawal, dead cells could be obSocs PROTEINS IN LIF-DEPENDENT ES CELL SURVIVAL

served (refringent cells in Fig. 1A), and a significant fraction of the DNA showed the classical degradation ladder (Fig. 1B) indicative of apoptotic chromatin damaging (37–39). The presence of apoptotic cells was confirmed by electron microscopy analysis (data not shown). After 5–7 days of LIF deprivation, the remaining cells started to morphologically differentiate and, concomitantly, the proportion of damaged DNA decreased to undetectable levels. A similar apoptotic process could also be observed on LIF withdrawal from a clonal cell line derived from the D3 cell line (40) (not shown). The apoptosis mediated by LIF deprivation apparently did not involve the specific caspases responsible for poly-ADP-ribose polymerase (PARP) cleavage (41), since this particular apoptotic marker was not cleaved in ES cells after LIF withdrawal (Fig. 2A, lane 2). However, treatment of these cells with the apoptogenic agent staurosporine led to PARP cleavage (Fig. 2A, lane 3), indicating that the PARP-specific caspases are indeed present and could be activated in ES cells. Also, we found that p38 MAP kinase isoforms were induced after 4 days of LIF withdrawal (Fig. 2B). The STAT3 protein level, which does not fluctuate in ES cells, is 1579

Figure 2. Apoptosis after LIF withdrawal is independent of PARP cleavage but involves activation of the p38 MAP kinase. A) Nuclear cell lysates (50 ␮g) from ES cells grown in the continuous presence of LIF (lane 1), in the absence of LIF for 4 days (lane 2), or in the presence of LIF with 100 nM staurosporine for 24 h, were analyzed with the anti-PARP antibody. The arrow points to the 113 kDa PARP protein. The asterisk indicates the position of the caspase-3 truncated PARP. B) Nuclear cell lysates (50 ␮g) from ES cells grown in the continuous presence (lane 1) or absence of LIF for 4 days (lane 2) or 8 days (lane 3) were analyzed with the anti phospho-p38 antibody that recognizes the phosphorylated ␣, ␦, and ␥ isoforms.

used here as an internal control of protein loading (17).

earlier that the STAT3 transcription factor is essential for LIF-dependent transcription (17). In this study, ES cells were cotransfected with this LIF-

Expression of potential LIF-responsive genes in ES cells By semiquantitative reverse transcriptase (RT) -PCR essay, we have analyzed expression of several genes such as 1) genes whose promoters contain STAT binding sites (like c-fos, junB, and socs-1; refs 42– 44) 2) genes known to be regulated during ES cell differentiation (like the ESP tyrosine phosphatase; ref 45), 3) genes induced by LIF in other cell systems (like c-jun and junD; ref 46), and 4) genes preventing apoptosis and exhibiting feedback repression activity on cytokine signaling (like the Socs genes; ref 30). In addition, we have characterized the expression of genes encoding the subunits of the LIF receptor (LIFr␤ and gp130). Total RNAs were prepared from ES cells grown for 20 h after LIF withdrawal (⫺ LIF), those grown for 20 h after LIF withdrawal and reinduced for 30 min with LIF (⫹ reinduction), or those maintained continuously in the presence of LIF (⫹ LIF). Transcripts corresponding to a ribosomal phosphoprotein (36B4) were used as an invariant internal control (34). As shown in Fig. 3, a clear induction of the c-fos, c-jun, junB, and Socs-3 transcripts was observed after LIF reinduction. High levels of Socs-3 mRNA also accumulated in ES cells continuously maintained in the presence of LIF. Under the same conditions, the mRNA levels of JunD, Socs-1, Socs-2, ESP, and the two major components of the LIF receptor (gp130 and LIFr␤) remained constant. Overexpression of Socs-1 and Socs-3 leads to repression of LIF-dependent transcription and cell lethality Overexpression of Socs proteins in different cell systems revealed their critical effect in the control of cytokine-dependent transcription (23, 47). Using a LIF-responsive (SIE)3-TK CAT reporter, we showed 1580

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Figure 3. Analysis by semiquantitative RT-PCR of LIF-induced genes. Total RNA (1 ␮g) from ES cells grown for 20 h after LIF withdrawal (⫺), grown for 20 h after LIF withdrawal and reinduced for 30 min with LIF, or maintained continuously in the presence of LIF (⫹) were analyzed by semiquantitative RT-PCR. The RT-PCR reactions (20 cycles) were migrated on a 1% agarose gel, blotted, and hybridized with labeled cDNAs as indicated on the left-hand side. The set of autoradiograms presented is representative of the results from at least two independent experiments, repeated with different RNA preparations.

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ti-HA antibody 48 h after transfection revealed similar levels of expression from each of these Socs constructs (data not shown). As Socs overexpression reduces LIF-dependent transcription, we tested whether overexpression of Socs-1 and Socs-3 affected ES cell pluripotency and/or growth properties. For this purpose, we attempted to derive stable ES cell clones overproducing either wild-type (S1WT, S3WT) or altered (S3⌬NC) Socs proteins. ES cells were cotransfected with the different Socs constructs, together with the PGK-neo selection vector. After 2 wk in selective medium, numerous neomycin-resistant clones were obtained with the control (V) and S3⌬NC vectors. By contrast, the number of surviving clones recovered with S1WT and S3WT plasmids was reduced by 80 to 90% (Table 1). These results clearly indicate that an excess of Socs-1 and of Socs-3 leads to cell lethality, most likely by blocking proper LIF signaling in ES cells.

DISCUSSION Figure 4. Overexpression of Socs-1 and Socs-3 represses LIF-dependent transcription. A) Wild-type Socs-1 or Socs-3 proteins (S1WT, S3WT) or the mutated Socs-3 proteins (S3⌬N, S3⌬C, and S3⌬NC) are represented with the positions of the conserved SH2 and Socs box domains. The coordinates of each domain were taken from (48). B) ES cells grown continuously in the presence of LIF (⫹) or 20 h after LIF withdrawal (⫺) were transfected with 5 ␮g of LIF-responsive [(SIE)3-TK CAT ] or 15 ␮g of LIF-unresponsive [(SIEm)3-TK CAT ] reporter constructs, together with 1 ␮g of the SV40based vectors, either empty (V) or expressing the HA-tagged Socs proteins, as indicated. About 36 h after transfection, the cells were collected and assayed for CAT activity. The mean values (⫾ sd) of four independent experiments are presented.

responsive [(SIE)3-TK CAT] or with a mutated, LIF-unresponsive [(SIEm)3-TK CAT ] reporter gene, together with vectors allowing overexpression of HA-tagged Socs-1 (S1WT) and Socs-3 (S3WT) and of mutated forms of Socs-3 in which the first 28 amino acids (S3⌬N), the last 50 amino acids (encompassing the Socs box) (S3⌬C), or both the amino- and carboxyl-terminal parts of the protein (S3⌬NC) were deleted (Fig. 4A). LIF-induced transcription from the (SIE)3-TK CAT reporter was impaired when Socs-1 and Socs-3, but not Socs-2, were overexpressed (Fig. 4B and data not shown). Repression of transcription was nearly abolished with S3⌬N, S3⌬C, and S3⌬NC, indicating that the integrity of both the Socs box and the first 28 amino acids of Socs-3 is required to efficiently block LIF-dependent transcription. No repression of transcription was detected with the (SIEm)3-TK CAT, indicating that Socs proteins abolished LIF-dependent transcription rather than basal transcription. Immunostaining of cells with the anSocs PROTEINS IN LIF-DEPENDENT ES CELL SURVIVAL

LIF prevents apoptosis in ES cells Apoptosis is a dynamic process that involves activation of caspases, MAP kinases, and particular endonucleases, leading to cell death (39, 41). Caspaseindependent programmed cell death has also been described (49, 50). We present evidence indicating that ES cells deprived of LIF enter an apoptotic crisis, a phase that precedes cell differentiation. A similar effect of LIF withdrawal has been reported in primordial germ cells and cardiac myocytes in which LIF is also essential for cell proliferation (51, 52). It is possible that in ES cells apoptotic signals are necessary to trigger ES cell differentiation by an as yet unknown mechanism. The threshold level of these signals might commit some of the cells toward irreversible apoptosis. Alternatively, one may imagine that some cells enter apoptosis and the remainder differentiate, depending on their stage in the cell cycle. The PARP, a survival factor essential in DNA-damaged cells, is specifically cleaved by caspase-3 during apoptosis, whereas inhibition of PARP cleavage delays apoptosis (41, 53, 54). The fact that PARP is not cleaved in ES cells after LIF withdrawal may retard apoptosis, allowing the differentiation program to take place. TABLE 1. Overexpression of Socs-1 and Socs-3 leads to cell death Expression vectors

neor clones

V

240

S1 WT

S3 WT

S3⌬NC

21

58

230

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Several observations suggest that STAT3 might prevent apoptosis in ES cells deprived of LIF: 1) the STAT3 transcription factor is rapidly inactivated after LIF withdrawal (17); 2) it prevents apoptosis in a proB-cell line and in T cells (55, 56); and 3) its constitutive activation blocks apoptotic processes in myeloma cells (57). Abrogating STAT3 function by different strategies induces morphological differentiation of ES cells despite a continuous supply of LIF (17, 20, 58, 59); it will be of interest to determine whether, as in the case of LIF withdrawal, part of the cells die by apoptosis during this differentiation process. We have shown that ERK1/ERK2 and JNK1 are activated on LIF treatment (17 and unpublished results). By contrast, p38 is activated after LIF withdrawal during the apoptotic crisis. Activation of p38 also occurs during neuronal apoptosis, after NGF withdrawal, and insulin-dependent cell survival has been shown to be linked to p38 repression (14, 15). Although the physiological significance of p38 activation remains to be established in our cell system, the possibility exists that it may be necessary for apoptosis and/or differentiation to take place. LIF-dependent transcription of early response genes Among the early response genes known to be activated transiently by cytokine treatment are the c-fos and JunB genes, which contain STAT binding sites in their promoters and whose expression correlates with LIFdependent STAT3 activation in ES cells (17, 60). Expression of these genes as well as c-jun is essential in LIF-responsive tissues (e.g., bones, liver; see ref 61, 62). Therefore, it is possible that expression of these genes in ES cells might prefigure their LIF dependency in organs in which LIF is a proliferative factor. LIF triggers opposite effects, depending on the nature of the target cells: it is rather proliferative in stem cell pools and induces differentiation in more committed progenitor cells (63). It is clear from previous studies with the myeloid M1 cell line and from our results in ES cells that induction of early LIF-responsive genes differs in cell systems in which LIF has opposite effects. Indeed, c-jun, junB, and c-fos are stimulated by LIF in both cell types whereas jun D expression, strongly induced during LIF-dependent differentiation of M1 cells (46), remains constant in ES cells (this work). Large-scale analysis of LIF-induced genes in different cell systems should lead to the identification of genes activated in differentiated compared to proliferative LIF-dependent pathways. This approach should help to characterize genes involved in the maintenance of cell totipotency. Socs-1 and Socs-3 proteins modulate LIF signaling in ES cells Socs-3 is strongly induced by short-term LIF treatment and highly expressed in ES cells continuously 1582

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maintained in the presence of LIF. STAT3 binding sites, necessary for LIF-mediated activation of transcription, have been characterized in socs-3 promoter (64). In contrast, socs-1 gene expression is not induced by LIF, although its promoter contains STAT binding sites (44). This indicates that STAT3 is probably not involved in socs-1 gene regulation in our cell system. The expression of socs-1 and socs-3 genes in ES cells differs with that reported in M1 cells in which Socs-1 is induced by LIF but not socs3 (27). However, forced expression of either socs-1 or socs-3 modulates LIF-dependent processes; it 1) represses STAT3-dependent transcription, 2) decreases ES cell growth, and 3) prevents M1 cell differentiation (27; this work). Socs-1 protein differs from Socs-3 in its amino-terminal proline-rich region, which may exhibit specific LIF-dependent SH3 domain recognition functions (28, 48). The molecular mechanism of repression of LIFdependent genes by the Socs-1 and Socs-3 proteins is presently unknown. The Socs box has previously been shown to interact with elongins B and C of the RNA polymerase II elongation complex (24, 25), raising the possibility that Socs proteins may act by modulating transcription elongation. It is striking that in ES cells, both the amino-terminal part and the carboxyl-terminal Socs box domains, of the Socs-3 protein are required to mediate the repressive activity, since like the double mutation, deletion of each domain separately abolishes the repressive effect. This observation suggests that the extremities of these molecules are functionally linked, with the Socs box interacting with part of the RNA polymerase II elongation machinery and the amino-terminal portion perhaps contributing to the specific targeting of LIF-responsive genes. The linker region located between the SH2 domain and the Socs box has recently been shown to be critical for the repressive effect of Socs-3 on growth hormone signaling (65). The possibility therefore exists that at least part of this linker domain may also contribute to the negative effect of Socs-3 on LIF-dependent transcription. As shown in the present study, overexpression of Socs-1 or Socs-3 in ES cells increases cell death. On the other hand, abolition of socs gene expression, as in socs-1 null mice, has been reported to generate apoptosis in liver and lymphoid organs (66, 67). These apparently divergent results might be explained by the disruption of a finely tuned balance between the levels of early response gene products and feedback regulators of LIF, whose relative concentrations must be critical for cell survival and for the maintenance of cell pluripotency. We thank A. Dierich, D. Queuche, and E. Blondelle for ES cells and expert advice, B. Chatton and M. Vigneron for helpful discussions and gifts of material, C. Wasylyk for DNA analysis procedure, and C. Hindelang for the electronic

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microscopy analysis of ES cells. We thank G. de Murcia for the gift of the anti-PARP antibody and N. Ghyselinck for critical reading of the manuscript. We are grateful to the staffs of the cell culture, biocomputing, and artwork facilities for providing help and material. This work was supported by funds from the Center National de la Recherche Scientifique, the Institut National de la Sante´ et de la Recherche Me´dicale, the Hoˆpital Universitaire de Strasbourg (H.V.S.), the Human Frontier Science Program, the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, and the Universite´ Louis Pasteur de Strasbourg.

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The FASEB Journal

Received for publication September 2, 1999. Revised for publication January 3, 2000.

DUVAL ET AL.