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Laura Schaefer,2 Suchithra Menon,1 Xing-Wang Deng,1 Jeffrey Boone Miller,2 and Ning Wei1*
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104,1 Neuromuscular Biology & Disease Group, Boston Biomedical Research Institute, Watertown, Massachusetts 024782
Received 25 April 2003/ Returned for modification 19 May 2003/ Accepted 27 June 2003
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ABSTRACT |
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B for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2-/- blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2-/- embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, and p53 in vitro (3, 35, 39). Phosphorylation of p53 by CSN-associated kinases at Thr155 promotes p53 degradation by the ubiquitin-proteasome pathway (3). Another target of the kinase activity is interferon consensus sequence binding protein, which is recruited to CSN through Csn2 (4). CSN interacts with SCF (Skp1-cullin-F-box protein) E3 ubiquitin ligases and deconjugates the ubiquitin-like protein Nedd8 from Cul1 (deneddylation) (5, 21, 34, 48, 50). This reaction counters the neddylation reaction catalyzed by the Nedd8-specific activating enzymes APP-BP1 and Uba3 (E1) and the Nedd8 conjugating enzyme Ubc12 (E2) (16). Neddylation has been shown to stimulate SCF ubiquitin ligase activities (17, 47). Disruption of Cul1, Cul3, or the neddylation enzyme Uba3 in mice leads to embryonic lethality and overaccumulation of an SCF substrate, cyclin E (6, 37, 43, 40). It has recently been shown that Cul1 deneddylation enables it to bind CAND1, which subsequently results in dissociation of Skp1-Skp2 from Cul1 (20, 49). As a result, both CSN deneddylase and CAND1 were shown to inhibit SCF activity in vitro (20, 48, 49). However, reducing the level of CAND1 in cells by small interfering RNA caused accumulation of p27kip1 (p27) (49). Similarly, genetic data indicate that a fully functional CSN5 is required for optimal activity of SCF in degradation of PSIAA6, a putative substrate of SCFTIR1 in Arabidopsis thaliana (34), and in degradation of Sic1 in Saccharomyces cerevisiae (5). This led to the hypothesis that both neddylation and deneddylation are necessary for the SCF functions.
CSN-mediated deneddylation is believed to center in the metalloprotease motif of CSN5/Jab1, but the manifestation of the activity requires the entire complex (5). Each of the null mutants of CSN subunits from Arabidopsis thaliana and fission yeast Schizosaccharomyces pombe lacks Cul1 deneddylation activity and preferentially accumulates the Nedd8 modified form of Cul1 (21, 34, 41, 50). In particular, Csn2 plays a critical role in deneddylation in part by direct binding to Cul1 and Cul2 primarily through its N-terminal region (amino acids 1 to 189), while it integrates into the CSN complex via its C-terminal region (amino acids 210 to 443) (48).
Csn2 was initially isolated as a thyroid hormone receptor-interacting protein (19) and was subsequently found to interact with a subset of additional nuclear hormone receptors such as DAX-1 (through human Alien/Csn2, amino acids 1 to 275), COUP-TF1, and ecdysone receptor (2, 9). Transient expression of a fragment corresponding to the N-terminal (amino acids 1 to 300) fragment of Csn2 Alien enhanced thyroid hormone receptor-mediated transcription repression, leading to the hypothesis that Csn2 represents a member of a new class of corepressors (9). The mechanism of this transcription cosuppression is unclear. Interestingly, forced expression of a full-length Csn2 was sufficient to induce neuronal differentiation and a concomitant decrease in Oct3/4 (Oct4) expression in P19 embryonal carcinoma cells in the absence of retinoic acid treatment (1).
The role of CSN in development has been studied only in nonmammalian genetic systems. CSN is involved in photomorphogenesis, flower development, and auxin and defense responses in plants (11, 34, 36, 42) and in oogenesis, embryogenesis, DNA repair, and axon guidance in flies (8, 12, 29, 38). CSN also plays an important role in the S-phase progression of the cell cycle in the fission yeast Schizosaccharomyces pombe (25). The CSN-like complex in the budding yeast Saccharomyces cerevisiae is involved in mating pheromone response (22). To understand the role of CSN in mouse development, we disrupted the murine Csn2 gene. Loss of Csn2 caused embryonic lethality at the peri-implantation stage and a deficiency of cell proliferation in the inner cell mass (ICM). The mutant embryos displayed elevated accumulation of cyclin E, p53, and the cyclin-dependent kinase inhibitor p21Cip1/Waf1.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Positive clones were karyotyped and subsequently injected into C57BL/6J blastocysts. Chimera males were mated with C57BL/6J females, and offspring were genotyped by PCR of tail DNA with primers ALA (5'-CCGCCGCTCGAGCTCTGCCTTTACAGTGTTCC) and En2 (5'-CCGCCGCTCGAGTACTTTCGGTTCCTCTTCCC) or the PCR primer sets used for blastocyst genotyping.
Blastocyst isolation, outgrowth, and genotyping. Blastocysts were collected at embryonic development day 3.5 (E3.5) by flushing the uterus with M2 medium (Specialty Media MR-015P-F). The blastocysts were then transferred to ES medium in a 0.1% gelatin-treated multiwell slidechamber slide (Lav-Tek, Nalgene) and cultured at 37°C with 5% CO2. After photographing or immunostaining, the blastocyst outgrowth colonies were lysed in buffer containing 10 mM Tris-HCl (pH 8.5), 50 mM KCl, 2 mM MgCl2, 0.45% (vol/vol) NP-40, 0.45% (vol/vol) Tween 20, and 60 µg of proteinase K per ml. The samples were boiled for 10 min to inactivate the protease. Half of the lysate (8 µl) was used in a 25-µl PCR, starting with denaturing at 94°C for 3 min, followed by 28 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 1 min. The PCR primers S2I2F (5'-GGGAACAGATCAATTGATTTCCTGA) and E3R4 (5'-GATCATCTGTTTTAGCGCTTTGAAT) were used to detect the wild-type Csn2 allele (252 bp). The primers S2I2F and En2 (5'-CCGCCGCTCGAGTACTTTCGGTTCCTCTTCCC) were used to detect the targeted allele (193 bp).
Immunohistochemistry. The pregnant uterus from timed heterozygous matings was surgically removed, and the individual decidua were separated and fixed in 3.7% formaldehyde (or 10% of the commercial 37% solution; J. T. Baker) overnight at 4°C. Samples were then embedded in paraffin blocks and cut into 5-µm sections. For antibody staining, the sections were deparaffinized and placed into a 3% solution of hydrogen peroxide for 5 min. After incubation with the primary antibodies for 1 h followed by washing, biotinylated secondary antibody was applied for 30 min and streptavidin-horseradish peroxidase was applied for 30 min. Diaminobenzidine was used as the peroxidase substrate, and counterstaining was performed with hematoxylin. The antibodies used include anti-CSN2 (1:1,000), anti-CSN1 (1:100), and anti-CSN8 (affinity purified, 0.7 mg/ml, at 1:250) (Affiniti Research Inc.), anti-cyclin E (M-20 Santa Cruz, 1:150), anti-p53 (Dako), and anti-p21 (C-19 Santa Cruz, 1:250).
Immunofluorescence staining and bromodeoxyuridine incorporation. Bromodeoxyuridine was added to culture media to a final concentration of 10 µM and incubated for 12 h at the time period specified. Cells were then fixed in 4% paraformaldehyde followed by DNA denaturation with 1.5 N HCl for 20 min. For other antibody stainings, the HCl treatment was replaced with permeablization solution (0.4% Triton X-100 in phosphate-buffered saline) for 10 min. The antibodies used include antibromodeoxyuridine (BD Biosciences-PharMingen); anti-Oct4 (Geneka, 1:30), and anti-Fgf4 (R&D Systems, 5 µg/ml). The fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies were obtained from Molecular Probes, Inc.
Inhibition of deneddylation by anti-CSN2 antibody in vitro. Inhibition of deneddylation by anti-CSN2 antibody in vitro was carried out as previously described (48). The total HeLa cell extract (40 µg) was supplied with ATP and ATP regeneration system and GST or GST-Nedd8 purified from Escherichia coli in amounts as indicated. Anti-Csn2 antiserum (1 µl) or preimmune serum (1 µl) was added and incubated at room temperature for 20 min. The reaction was terminated by adding sodium dodecyl sulfate loading buffer. The antibodies used for the immunoblots include anti-Cul1 (1:1,000, H-213, Santa Cruz,), anti-Cul2 (1: 250, Zymed), and anti-glutathione S-transferase (GST) (1:1,000, Pharmacia).
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RESULTS |
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Csn2-/- embryos arrest at peri-implantation stage. The Csn2+/- heterozygous mice appeared phenotypically healthy and fertile at up to 1 year of age. The Csn2 expression pattern in embryos, as indicated by LacZ staining of the heterozygous embryos, resembled that revealed by in situ hybridization reported previously (33). These heterozygous mice were intercrossed to produce Csn2-/- mice. However, no nullizygous mice were found among over 86 offspring, indicating that loss of Csn2 caused recessive embryonic lethality. Embryos were isolated from timed Csn2 heterozygous intercrosses from embryonic day 11.5 to as early as E7.5 and genotyped. As shown in Table 1, no viable Csn2 -/- embryos were detected at any of the postimplantation stages, but abnormally high numbers (more than a quarter of total embryos) of empty decidua were observed. We next isolated and genotyped preimplantation stage embryos (blastocysts) at E3.5 and found that Csn2 -/- blastocysts amounted to 16% of the total (Table 1). This result indicates that Csn2-/- embryos survived to blastocyst stage, underwent implantation as the mutants elicited decidual swelling, but died soon after implantation.
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Csn2 is required for cellular proliferation of the inner cell mass. The newly isolated Csn2-/- blastocysts (day 0) were viable and largely indistinguishable from those of the wild type. These blastocysts were cultured in vitro to allow outgrowth for 5 days (Fig. 3A) and were then genotyped (Fig. 3C). Both Csn2+ and Csn2-/- blastocysts hatched from the zona pellucida (day 1), attached onto the culture dish (day 2). Although the Csn2-/- blastocysts seemed to be more sensitive to culture conditions, they can produce apparently normal trophoblast giant cells, a process necessary to induce the decidual reaction during implantation. However, the inner cell mass (ICM), which forms the future embryonic tissues, failed to outgrow in Csn2-/- blastocysts (Fig. 3A).
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The ICM failure prompted us to examine the expression of Oct4, a POU domain transcription factor required for establishing and maintaining ICM and other stem cells. The precise level of Oct4 is critical for proper cell differentiation and development in early embryos (28). Disruption of Oct4 results in the diversion of ICM cells into the trophectoderm cell lineage, leading to early embryonic lethality (27). In the Csn2-/- blastocysts just hatched from the zona (day 1), the ICM-specific expression pattern of Oct4 was largely maintained in the mutant, but the level was slightly reduced (Fig. 3D-b). We also examined an Oct4 downstream target, fibroblast growth factor 4 (Fgf4), whose expression is critical for postimplantation embryo development (10). Fgf4 expression pattern appeared slightly altered in that its level was detectably decreased in the presumptive ICM cells but slightly increased in the trophectoderm cells (Fig. 3D-d). These results indicate that Csn2 is necessary for precise expression of Oct4 pathway components in early embryos.
Loss of Csn2 leads to elevated levels of cyclin E. Csn2 plays a critical role in CSN-mediated deneddylation. Incubation of the anti-Csn2 antibody in HeLa cell extract decreased the proportion of the unneddylated Cul1 and Cul2 with concomitant increase in the neddylated forms (Fig. 4A) (48, 49). This is caused by selective blockage of deneddylation in the extract that was active in the neddylation reactions as confirmed by de novo conjugation of exogenously supplied GST-Nedd8 in the assay (Fig. 4A). Conjugation of GST-Nedd8 appeared less efficient in the presence of anti-Csn2 (Fig. 4A), probably due to steric hindrance of the antibody when bound to CSN-SCF tertiary complex. This result demonstrates that cycles of neddylation and deneddylation are highly dynamic even in the cell extract, and it emphasizes the important role of Csn2 in deneddylation. Although we were unable to directly demonstrate a deneddylation defect in Csn2-/- cells due to early lethality of the mutant, this result combined with the observations that all null csn mutants from other genetic systems lack Cul1 deneddylation activity strongly argue that inactivation of Csn2 would almost certainly lead to inhibition of CSN deneddylase activity.
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Csn2-/- cells contain higher levels of p53 and p21. The tumor suppressor p53 is thought to promote genomic stability, induce growth arrest and apoptosis, and consequently inhibit tumorigenesis. The protein turnover of p53 is tightly controlled in the cell through multiple E3 ubiquitin ligases. In addition, p53 interacts with CSN5/Jab1 and is a phosphorylation substrate of CSN-associated protein kinase (3). We detected p53 expression only in extraembryonic endoderm cells of normal E6.5 embryos (indicated by an arrow, Fig. 5A). In Csn2-/- embryos, in contrast, p53 was intensely stained in essentially all of the mutant cells, including the presumed inner layer cells at E6.5 (Fig. 5B and 5C). Moreover, in vitro-cultured Csn2-/- blastocysts contained strong anti-p53 immunoreactions in the presumed ICM cells compared to the Csn+ blastocyst outgrowth (Fig. 5D and 5E). These data indicate that there is a general increase in p53 level in the mutant cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recent report showed that enforced expression of Csn2 caused downregulation of Oct4 and was sufficient to convert P19 cells into neurons but not glial cells, and that antisense Csn2 suppressed neuronal differentiation (1). Our data indicated that the arrest of Csn2-/- embryos at implantation stage was primarily caused by deficient ICM cell proliferation accompanied by a decline in Oct4 level. In addition, since differentiation of the presumed ICM cells was not detected in the mutant embryos (Fig. 2), this implies that Csn2 is required for both cell proliferation and differentiation during early embryo development.
One of the major activities of CSN is to deneddylate SCF Cul1. Neddylation of SCF has been shown to stimulate the assembly of polyubiquitin chains and the recruitment of an E2 (17, 47). It is also required for ubiquitination of specific SCF substrates such as p27 and IB (13, 23, 30, 31). Even though the deneddylation pathway components, CSN and CAND1, appear to inhibit SCF activity in vitro (20, 48, 49), our genetic data support the idea that CSN ultimately facilitates SCF activity in vivo. Accordingly, both neddylation and deneddylation seem necessary for proper SCF function. Components of the SCF complex, such as Cul1 and Skp2, have been shown to be ubiquitination targets of its own E3 activity in vitro and in vivo (24, 46, 51). It is conceivable that a mechanism restricting the self-ubiquitination activity is necessary to maintain the sustained SCF activity in vivo. We hypothesize that the control of SCF activation-inactivation may be achieved through the neddylation-deneddylation cycle. This is supported by the finding that knockdown of CAND1 by small interfering RNA decreased the level of Skp2, a substrate receptor for SCF, resulting in stabilization of p27 (49). Taken together, our data strongly support the notion that the dynamic cycles of neddylation and deneddylation are necessary for the optimal SCF functions in vivo. Nevertheless, we cannot exclude the possibility that other activities of CSN such as the associated protein kinase activity, which potentially affects substrate phosphorylation and thereby recruitment to the ubiquitin ligases, may be responsible for accumulation of SCF substrate such as cyclin E in the Csn2-/- mutant.
Our finding that p53 highly accumulates in Csn2-deficient cells is consistent with the previous report that CSN interacts with and phosphorylates p53, leading to ubiquitin-proteasome-dependent degradation (3). Whether the abnormal accumulation of p53 is caused by defects in CSN-mediated phosphorylation or in its ubiquitination through Mdm2-p300, Pirh2, or Cul5 complexes or by other means requires further study. It is likely that the increased abundance of p53 and elevated expression of p21Cip1/Waf1 in the Csn2-/- embryos are directly relevant to its premature cell cycle arrest and possibly cell senescence. Certainly, other defects caused by CSN deficiency, including decreased expression of Oct4, may also contribute to the early embryonic phenotype. Interestingly, microinjection of the purified CSN complex has been shown to cause cell cycle delay (48). Thus, similar to the scenario of neddylation and deneddylation with regard to SCF function, either gain of function or loss of function of CSN can lead to perturbation of cell cycle progression.
The CSN complex and Csn2 have been implicated in nuclear hormone receptor-mediated gene expression, stress-activated protein kinase cascade, and cell cycle control in mammalian cells (1, 2, 26, 48). In nonmammalian eukaryotic organisms, CSN participates in a variety of developmental processes. We demonstrate here that Csn2 and the CSN complex are essential for ICM cell proliferation and early embryonic development in mice, underscoring the fundamental role of this conserved protein complex in development and in cellular homeostasis.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants to N.W. from NIH (GM61812) and EntreMed, Inc., and by grants to J.B.M. from NIH (AR49496, ES11384, HL64641), USDA, and the Muscular Dystrophy Association L.S. was supported by a postdoctoral fellowship from NIAMS.
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FOOTNOTES |
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Present address: The Wellcome Trust and Cancer Research UK Institute, University of Cambridge, Cambridge CB2 1QR, United Kingdom.
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