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Previous Article | Next Article 
Molecular and Cellular Biology, October 2003, p. 6798-6808, Vol. 23, No. 19
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.19.6798-6808.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Hannes Vogel,2 Ning Wei,3 Monica J. Justice,1 Allan Bradley,4 and James R. Lupski1,5,6*
Department of Molecular and Human Genetics,1 Department of Pediatrics, Baylor College of Medicine,5 Texas Children's Hospital, Houston, Texas,6 Department of Pathology, Stanford University, Stanford, California,2 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut,3 Sanger Centre, Cambridge, United Kingdom4
Received 17 April 2003/ Returned for modification 19 May 2003/ Accepted 27 June 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3-Mb inversion chromosome. The heterozygous mice appeared normal, although the protein level was reduced. Csn3-/- embryos arrested after 5.5 days postcoitum (dpc) and resorbed by 8.5 dpc. Mutant embryos form an abnormal egg cylinder which does not gastrulate. They have reduced numbers of epiblast cells, mainly due to increased cell death. In the Csn3-/- mice, subunit 8 of the COP9 complex was not detected by immunohistochemical techniques, suggesting that the absence of Csn3 may disrupt the entire COP9 complex. Therefore, Csn3 is important for maintaining the integrity of the COP9 signalosome and is crucial to maintain the survival of epiblast cells and thus the development of the postimplantation embryo in mice. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations in the genes encoding different subunits convey common phenotypic features. However, they can also cause distinct phenotypes, indicating that each subunit may have unique functions. D. melanogaster mutants carrying null mutations of csn4 and csn5 both displayed defects in oogenesis, embryo patterning, and larval lethality, but csn4 null flies had molting defects, while csn5 null flies developed melanotic tumors (30). Distinct functional roles for different subunits have also been observed in Schizosaccharomyces pombe (29). Reduction-of-function mutations of the Arabidopsis thaliana CSN3 gene decreased the COP9 complex level and caused defects in diverse aspects of plant development, which indicates the involvement of CSN in multifaceted developmental processes (31). The function of CSN in mammals and how alteration of CSN and subunit 3 will affect mammalian development remain unknown.
Human CSN3 maps to the Smith-Magenis syndrome common deletion interval (8, 13, 32). Smith-Magenis syndrome is a multiple congenital anomaly/mental retardation syndrome associated with developmental delay, anatomical developmental defects, and neurobehavioral abnormalities associated with del(17)(p11.2p11.2) (16). The mouse Csn3 homolog resides in the region on chromosome 11 syntenic to the human Smith-Magenis syndrome deletion interval (4, 34). We disrupted Csn3 in mice to investigate its role in mammalian development and in the Smith-Magenis syndrome phenotypes. Homozygous mice arrested after 5.5 days postcoitum (dpc) showed abnormalities in egg cylinder formation and failure of development at the gastrulation stage. Also, the CSN complex appeared to have been disrupted. This indicates the essential role of CSN in mammalian early embryonic development.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess Csn3 gene expression, a 1.7-kb fragment containing the Csn3 full-length cDNA was isolated from the Invitrogen Pcops3 clone with BamHI and ApaI and used to probe both the adult and embryonic mouse multiple tissue Northern blot (Clontech) according to the method provided by the manufacturer.
Construction of Csn3 targeting vectors. Mouse strain 129/SvEv genomic libraries constructed in vectors containing either the 5' half of the human HPRT minigene (5' HPRT vector) or 3' half of the gene (3' HPRT vector) were screened (48) to identify both 5' and 3' HPRT insertional vectors. One clone, pWY1-3', from the 3' HPRT library has an 8-kb genomic fragment of Csn3 and contains exons 3, 4, 5, and 6, as determined by restriction mapping and PCR analysis. The pWY1-3' clone was digested with BglII and NdeI to delete a 3-kb fragment corresponding to exon 5 and adjacent intronic regions. After fill-in repair with Klenow fragment polymerase and subsequent religation, a BglII site was reconstructed, and this site was used to linearize the vector before introduction into embryonic stem (ES) cells. An NdeI linker was ligated into the reconstructed BglII site, and then the genomic fragment was isolated from the 3' HPRT targeting vector with AscI and placed into the 5' HPRT vector to get the 5' HPRT targeting vector pWY1-5'. This NdeI site was used as a linearization site for pWY1-5'.
Targeting in embryonic stem cells and germ line transmission. The linearized vectors pWY1-5' and pWY1-3' were introduced into the AB2.2 ES cells derived from Hprt-deficient 129SvEvBrd mice (28). ES cell growth, electroporation, and drug selection were performed as described previously (28, 35). The chimeric mice were mated to C57BL/6 wild-type mice, and F1 progeny were back-crossed to C57BL/6 mice to produce N2 and N3 mice. Two ES cell clones with the 5' HPRT vector pWY1-5' and one clone with the 3' HPRT vector pWY1-3' were transmitted through the germ line. Mouse strains targeted with the pWY1-5' vector and pWY1-3' vector are referred to as the Csn35'm strain and the Csn33'm strain, respectively, and the targeted alleles are referred to as the Csn35'm and Csn33'm alleles, respectively. Both strains were maintained by backcrossing to C57BL/6 wild-type mice. All the animals were treated in compliance with relevant animal welfare policies.
Confirmation of targeting.
After drug selection, targeted ES cells were detected by diagnostic Southern analysis with NdeI digestion. The probe was a 550-bp PCR fragment to the region that was deleted from the targeting vector by BglII and NdeI amplified with primers E5PR (5'-TCC CAT TGT AAG CCC CAC TA-3') and E5PF (5'-GCA ATA CGG TTT ATT AGT GAT AGC C-3'). The PCR product was gel purified (Qiagen gel extraction kit) and labeled with [
-32P]dCTP by random priming (Amersham Rediprime II).
Targeted clones revealed an anticipated 18-kb fragment in addition to the 6.5-kb wild-type fragment. The targeted F1, N2, and N3 mice were confirmed by PCR and/or Southern analysis. One primer complementary to the targeting vector near the genomic fragment insertion site was synthesized, and the other primer was constructed to the genomic fragment. For the 5' HPRT targeting vector, primers 5' HPRT ex3-psk (pSK vector backbone) F (5'-GAC ATG TGT GCG TCT CCA TA 3') and 5' HPRT 2ex6-psk R (5'-CCT GAT TCT GTG GAT AAC CGT ATT A-3') were used to yield an 800-bp product; Primers 5' HPRT 2ex6-psk F (5'-GAC ATT CTC AGG AGC TCA GTA GGT A-3') and 3' HPRT ex3-psk R (5'-CGA GAA AGG AAG GGA AGA AAG-3') are for the 3' HPRT targeting vector, and PCR amplification with them yields a 350-bp product. Both primers are unique for their own targeting vector and do not amplify from the wild-type DNA. Primers for the Myo15 gene, Myo15 F (2) (5'-CTC TAC AAG CAC CTG CCC TC-3') and Myo15 R (2) (5'-TAT TTC TCA AAG CTG TCA CTT TGC-3') were used as a positive control for DNA quality. EcoRI was used in Southern analysis with the same probe used for ES cell genotyping. This probe revealed an expected 12-kb wild-type band, a 10-kb band in Csn35'm strain mice, and an 18-kb band in Csn33'm strain mice.
Construction of a 3-Mb Csn3-Zfp179 chromosomal inversion. Zfp179 was formerly targeted with a vector from the 5' HPRT genomic library containing a genomic fragment with all the Zfp179 exons except for the last exon. Csn3 was subsequently targeted with pWY1-3' vector in the ES cell clone with targeted Zfp179 (42). Cre recombinase was introduced into double-targeted ES cell clones. After sodium hypoxanthine, aminopterin, the thymidine selection, inversion clones were confirmed by SacI digestion to yield the expected three bands at 28, 12.5, and 10 kb on diagnostic Southern blot analysis. The ES cell clone with the Csn3-Zfp179 chromosomal inversion was injected to produce inversion mice. Homozygous inversion mice are predicted to lose the 10-kb wild-type fragment on Southern blot analysis.
Western blotting analysis. Proteins were extracted from the livers of wild-type and Csn35'm/+ or Csn33'm/+ littermates with Trizol following the manufacturer's instructions (Invitrogen). Total protein (10 µg) was electrophoresed on sodium dodecyl sulfate-4 to 20% polyacrylamide gels (ISC) and transferred to a nitrocellulose membrane. Anti-mouse Csn3 polyclonal antibody directed against the entire Csn3 protein was produced in a rabbit. After incubation with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Bio-Rad) and enzymatic reaction (Santa Cruz ECL system), blots were developed, and the signal was quantitated with Un-scan-it software (Silk Scientific). Antiactin antibody (Santa Cruz) was used as a loading control.
Genotyping of homozygous embryos. BspHI was used to discriminate the heterozygous and the homozygous mice from the Csn35'm strain F1 or N2 heterozygous intercrosses with the same probe from the exon 5 described above. AseI was used for the Csn33'm strain N2 heterozygous mice intercrosses and EcoRI and PCR primers for genotyping the Csn35'm strain and Csn33'm strain were used for Csn35'm/+ and Csn33'm/+ intercrosses. DNA from 9.5 to 11.5 dpc embryos was used for genomic Southern analysis. Lysis buffer (100 µl: 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-Cl, pH 8.4, 0.5% Tween 20) was added to the yolk sac from the 9.5 dpc to 11.5 dpc embryos. After incubation at 55°C for an hour, proteinase K (1 mg/ml) (Roche) was inactivated by heating at 100°C for 15 min, and 1 or 2 µl was used for PCR analysis. For 8.5 dpc embryos, the whole embryos were lysed by adding 100 µl of lysis buffer, and 2 to 3 µl was used for PCR. For 7.5 dpc embryos, 15 µl of lysis buffer was added, and 2 to 4 µl was used for PCR. All the embryos analyzed, including those for histological analysis, were products of conception from Csn35'm/+ and Csn33'm/+ intercrosses.
Histological analysis of embryos. Embryos at 5.5 dpc to 7.5 dpc were fixed in 4% paraformaldehyde in 1x phosphate-buffered saline (PBS) at 4°C for 4 h to overnight, embedded in paraffin, and sectioned at a thickness of 6 to 7 µm. Sections were stained with hematoxylin and eosin. Immunohistologic analysis was carried out for the selected sections. After rehydration, sections were incubated with 10% goat serum in PBS at room temperature for 1 h, followed by incubation with primary antibody (anti-Csn3 antibody or anti-Csn8 antibody) and peroxidase-conjugated secondary antibody (Vector ABC kit) in PBS with 2% goat serum at room temperature for 1 h each. Color reaction was implemented following the manufacturer's recommendation (Vector, DAB kit). These sections were counterstained with hematoxylin.
Ice-cold methanol (100%) was used to fix the 3.5 dpc blastocysts. Blastocysts were then processed through 75%, 50%, and 25% methanol in PBST (1x PBS with 0.1% Tween 20), followed by washing once in PBST. Whole-mount immunostaining was carried out as for the sections except that tetramethyl rhodamine isothiocyanate-conjugated secondary antibody was used (Sigma). A Zeiss Axioplan2 fluorescence microscope was used to detect the signal. DNA for blastocyst genotyping was prepared by adding 10µl of 1x PCR buffer (Qiagen, HotStarTaq) to the blastocyst, incubating at 95°C for 20 min, and freezing (-80°C) for 1 h, and 5 µl was used for the PCR. Primers 5' HPRT 2Ty (tyrosinase gene) F (5'-CTG GGA GAA AAC ATA TTT TGA GAG A-3') and 5' HPRT TyR (5'-CCA CGA ATG CTG ACA TTC TC-3') were used for 5' HPRT targeting vector instead of those mentioned above.
In situ hybridization of whole-mount embryos. A probe for the Brachyury gene was prepared as described previously (19). In situ hybridization was carried out for two litters of 7.5 dpc embryos with Wilkinson's protocol (45) with some modifications: the proteinase K treatment step was omitted, and 4% paraformaldehyde was used for all the fixation steps. After photography, embryos were processed for PCR genotyping. Primers were the same as for genotyping the blastocysts.
TUNEL analysis. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of the selected sections was carried out according to the manufacturer's instructions (Roche, fluorescence cell death detection kit). The cells were visualized under a Zeiss Axioplan2 fluorescence microscope.
Bromodeoxyuridine incorporation analysis. Pregnant females were injected intraperitoneally with bromodeoxyuridine (100 µg/g of body weight) and sacrificed 60 to 70 min later. Embryos were processed for histology. Bromodeoxyuridine was detected following the manufacturer's protocol (Roche, bromodeoxyuridine labeling and detection kit II) except that after dewaxing and rehydration, sections were incubated with 0.02% pepsin (Sigma) in 0.01 N HCl for 20 min at 37°C.
Blastocyst outgrowth studies. Blastocysts at 3.5 dpc were cultured in ES cell medium (containing no leukemia inhibitory factor) in 5% CO2 at 37°C. The blastocysts were photographed after 3 days and after either 5 or 6 days in culture. The cultured embryos were processed for PCR as for 7.5 dpc embryos.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3.4 kb may represent an alternative splicing product or cross-reactivity.
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Two alleles were constructed to enable PCR genotyping of early embryos. After deletion of a 3-kb fragment corresponding to exon 5 and adjacent intronic regions with BglII and NdeI, both vectors were introduced into AB2.2 ES cells (28). Homologous recombination is predicted to result in the repair of the deleted region around exon 5 (18), duplication of exons 3, 4, 5, and 6, and the insertion of the 10-kb vector (17). Such rearrangements were confirmed by PCR and Southern analysis of targeted ES cells. Because of the insertion of the 10-kb vector, the transcription of the gene should be disrupted after exon 6. Even if the Csn3 transcript can be made by skipping the entire vector, by conceptual translation, duplication of exon 3 results in a frameshift and a stop codon immediately after the first exon 6. As expected, no truncated protein was observed by Western blot analysis in the heterozygous mice with Csn3 antibody directed against the entire protein, suggesting that the truncated protein probably was not formed or was immediately degraded or that the message was degraded by nonsense-mediated decay.
Targeted ES cell clones are anticipated to have an 18-kb fragment in addition to the 6.5-kb wild-type fragment after NdeI digestion and visualized with a probe from exon 5 on diagnostic Southern blot analysis (Fig. 1). Of the 192 targeted ES cell clones screened with each targeting vector, about 20% of the clones have the expected pattern on Southern blots consistent with the predicted targeting event. Three such clones, two from 5' HPRT vector targeting and one from 3' HPRT vector targeting, were injected into the blastocysts from C57BL/6 mice. Chimeras were produced and germ line transmission was established. F1 mice from the chimeras were backcrossed to C57BL/6 mice to obtain N2 and N3 progeny mice. The targeted allele was confirmed in mice by Southern analysis with EcoRI, which yielded the predicted 10-kb band in the 5' HPRT vector targeted allele (Csn35'm allele) and an 18-kb band in 3' HPRT vector targeted allele (Csn33'm allele) in addition to the 12-kb wild-type allele and/or by PCR analysis (800 bp for Csn35'm allele, 350 bp for Csn33'm allele).
Disruption of Csn3 by chromosome inversion. Zfp179 (47) was formerly targeted with an insertional vector from the 5' HPRT library (48), which resulted in the duplication of the entire gene except for the last exon. Csn3 was subsequently targeted with the 3' HPRT targeting vector in the ES cell clone containing the targeted Zfp179 gene (42). After introduction of the Cre recombinase, the entire chromosomal region between Csn3 and Zfp179 was inverted, yielding animals harboring an inv(11)17(Csn3-Zfp179) chromosome, which disrupted the Csn3 gene after exon 6 and retained the duplication of Zfp179 (Fig. 3). The inversion was confirmed by diagnostic Southern blot analysis with SacI digestion and a probe from Zfp179. Heterozygous inversion mice appeared normal. Homozygous inversion mice were absent in 32 offspring from intercrosses of the heterozygous inversion mice (Table 1), indicating embryonic lethality of the homozygous inversion. Since Zfp179 homozygous duplication mice showed no phenotype (data not shown), homozygous disruption of Csn3 most probably caused the lethality.
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Phenotype of Csn3-/- mice in early embryogenesis. To determine the time of developmental arrest during embryogenesis, timed matings were performed with crosses between Csn35'm/+ and Csn33'm/+ mice (Table 2; Fig. 5). From 9.5 to 11.5 dpc, 41 embryos were examined, 29 of which (71%) were morphologically normal. Of these 29 embryos, 21 were heterozygous and were indistinguishable from the wild-type littermates. No homozygous embryos were observed. The remaining 12 embryos were being resorbed at these stages, and DNA could not be recovered for genotyping. At 8.5 dpc, 8 out of 31 embryos examined were abnormal, 7 of which were starting to be resorbed. One Csn3-/- homozygous embryo was detected which had developed a head anlage, but this was much smaller than normal. It appeared transparent and did not have other structures that should have developed at that time (e.g., somites) (data not shown). Since we only observed one such embryo, it probably represents an exception. Twenty-three 7.5 dpc embryos were examined. Seven were homozygous, and each was one quarter to one fifth of the size of the normal embryos. They had an elongated shape similar to the normal embryos but appeared to be arresting prior to gastrulation (Fig. 5A and B). Therefore, Csn3-/- embryos arrested development before 8.5 dpc and were mostly resorbed by 8.5 dpc.
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In order to assess the effects of homozygous disruption of Csn3 on preimplantation embryos, immunohistochemistry with anti-Csn3 antibody was performed on 3.5 dpc blastocysts. Fourteen blastocysts were stained, two of which were confirmed to be mutants by PCR (data not shown). The mutants were normal in morphology, which was expected because there was only a slight difference between the mutants and normal embryos at 5.5 dpc. The Csn3 staining was indistinguishable between the mutant and the wild-type blastocysts, consistent with maternal protein deposition in the blastocysts.
A distinct and obvious mesoderm layer was apparently not formed in Csn3-/- embryos, but the disorganized epiblast cells may have obscured the discrimination of mesodermal cells. In order to examine mesoderm development in the mutants, the expression pattern of the Brachyury gene was evaluated. Brachyury is one of the earliest markers for mesodermal cells. At 7.5 dpc it is expressed in the primitive streak, in which the mesoderm formation begins, in the node, and in the notochordal plate extending anteriorly from the node (9, 19). Normal embryos showed the expected expression pattern, whereas the Csn3-/- mutant embryos showed no Brachyury expression (Fig. 6). This indicates that the primitive streak and the mesoderm were not formed in the Csn3-/- embryos, consistent with our histological observations. In summary, Csn3-/- mice arrested at early embryonic development after 5.5 dpc and fail to undergo gastrulation.
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Blastocysts outgrowth studies. As an alternative approach to investigate postimplantation development, we examined blastocyst outgrowth in vitro. In total, 11 blastocysts were examined, of which two were documented to be Csn3-/- mutants by genotyping. The blastocysts of the mutants were morphologically normal (Fig. 8). After 3 days of culture, the inner cell mass (ICM) growth of the mutants was comparable to that of the normal littermates. The number of ICM cells was greatly reduced after 5 to 6 days of culture in the mutants. The trophoblastic cells grew similarly in both mutant and normal embryos. It appears that the ICM initially developed and then there was reduced cell growth. These findings are consistent with our in vivo observations that epiblast cells proliferate normally until 6.5 dpc in Csn3-/- embryos, but the cell number is reduced secondary to cell death.
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DISCUSSION |
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In D. melanogaster, maternal Csn4 or Csn5 protein deposited in the csn4 or csn5 null embryos supported embryonic development until the late larval stage. Without maternally contributed proteins, oogenesis was arrested (30). In mice, mutant embryos can grow with maternal deposition (40), but maternal RNAs are usually degraded by 6 dpc (36), and embryos after that stage require at least some de novo gene expression (20). In Csn3-/- blastocysts, the Csn3 staining was similar to that in the normal embryos, which indicates that there is maternal protein deposition. At 5.5 dpc, the Csn3 protein level was greatly reduced, Csn3-/- showed slight defects at 5.5 dpc and grew through 6.5 and 7.5 dpc. It is difficult to determine whether the survival to this stage can be entirely attributed to a maternal effect or whether the Csn3 protein requirements for development are less before 6.5 dpc because of the slower rate of cell proliferation compared to that after 6.5 dpc, or perhaps a combination of both possibilities.
Two biochemical functions have been found for the CSN. One is the protein kinase function, and the other is controlling the E3 ubiquitin ligases, through which CSN controls the degradation of a wide variety of proteins, including those involved in the cell signaling pathway, such as c-Jun, and those in cell cycle control, such as p53 and p27kip1 (3, 37, 41). Microinjection of the CSN complex into HeLa cells inhibited p27kip1 degradation and impeded G1-S phase progression (46). Obviously, CSN controls cell growth and proliferation, but how disruption of the complex will affect cells is largely unknown.
In Csn3-/- mice, bromodeoxyuridine incorporation was similar to that in wild-type mice. However, cell death appeared to play a major role in the developmental arrest of the Csn3-/- mice. The cell death may be explained by the accumulation of molecules involved in the apoptotic pathway such as p53 or the reduction of certain factors important for the survival of the cell. Recently, it was demonstrated that Csn5 is important for the repair of DNA double-strand breaks, and its absence during meiosis activates a DNA damage checkpoint in D. melanogaster (12). In mammalian cells, p53 responds to DNA damage by delaying the cell cycle or inducing programmed cell death (22, 24). To elucidate whether the cell death in Csn3-/- mice is induced by double-strand breaks requires a further understanding of CSN function. Epiblast cells undergo rapid cell division just prior to gastrulation, which may explain why they were primarily affected.
So far, many genes have been found to be important for early embryonic development. Fliih and Cgbp were essential for development at implantation (4.5 dpc) (5, 6). Hnf-4 homozygous mice showed defects at 7.5 dpc (9). The phenotypes of our mutants are more similar to those of Rad51 homozygous knockout mice, which also start to show defects at 5.5 dpc and are resorbed by 8.5 dpc (26). Both mutants formed the proamniotic cavity, failed to go through gastrulation, and had increased cell death in the epiblast. However, decreased cell proliferation was more obvious in Rad51-/- embryos than in our Csn3-/- animals. Interestingly, mouse Rad51 is homologous to Rad51 in Saccharomyces cerevisiae, which repairs double-strand breaks by recombination. Mouse Rad51 may also be associated with double-strand break repair because trophectoderm-derived cells from homozygous mutants were sensitive to gamma radiation, and the mutant embryos lived longer and developed further in a p53 mutant background. It will be interesting to see if there is any relation between CSN and Rad51.
In Arabidopsis thaliana, mutations in most of the CSN subunits, including CSN3, can lead to the failure of CSN complex formation (31, 44). CSN3 has previously been demonstrated to interact directly with CSN8 (15). In our case, Csn8 was undetectable in the Csn3-/- embryos, suggesting that the entire complex was disrupted. As a result, at least certain functions of the complex, such as the SCF deneddylation activity that requires the entire complex (10), were most likely to be deficient in the Csn3-/- cells. Csn4 and Csn5 were both found not only in the CSN complex but also in other forms in D. melanogaster, and in csn5 null mutants the complex was formed (23, 30). It appears that some of the subunits have their own functions independent of the CSN complex. Overexpression of Csn5 promoted the degradation of p27kip1 in mammalian cells (41), while high levels of CSN complex inhibited p27kip1 degradation (46). The human CSN3 was also found in two to three larger complexes than the CSN complex (38). It is unknown if those larger complexes include CSN. It is likely that the phenotype observed in the Csn3-/- mice resulted from the disruption of both the CSN complex and the Csn3-specific functions.
In addition to an important role in early embryonic development, Csn3 may be required in later development and in different tissue functions. Reduction-of-function lines of CSN3 in A. thaliana showed diverse developmental defects (31), and Northern analysis showed ubiquitous expression in adult tissues of mice. To elucidate the role played by Csn3 in later development and in different organ systems, conditional knockout mice will be required.
Smith-Magenis syndrome is caused by a microdeletion on 17p11.2, and clinical manifestations include mental retardation, skeletal abnormalities, sleep disorder, and neurobehavioral anomalies (16). The phenotype is thought to result from the haploinsufficiency of one or more genes. Since heterozygous Csn3 knockout mice showed no obvious phenotypes, it may not be the gene responsible for the Smith-Magenis syndrome phenotypes. Previously, Csn3 was speculated to be possibly responsible for the sleep disorder because it controls light-mediated development in plants (33). Sleep studies in heterozygous Df(11) (Csn3-Zfp179) mice (42) are ongoing.
Another important reagent developed in our study was an inv(11)17(Csn3-Zfp179) inversion chromosome. Balancer chromosomes are used widely in D. melanogaster for stock maintenance and mutagenesis screens (2). Recently, a balancer chromosome was introduced into mouse genetics and used for a large-scale mouse mutagenesis screen (21, 49). The 3-Mb inversion covering the Smith-Magenis syndrome mouse syntenic region may be used to maintain individual mutations in genes that lie within this interval.
In conclusion, we demonstrated the importance of Csn3 in CSN complex assembly and its essential role in early mouse embryonic development. It will be interesting to investigate further the potential relationships between CSN and cell death and CSN and double-strand break repair and other molecules involved in double-strand break response and repair, such as Rad51. This may enable new insight into CSN function.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from the National Cancer Institute, NIH (PO1CA75719).
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FOOTNOTES |
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Present address: Celera Genomics, Rockville, MD 20850.
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