- Email: [email protected]
CDC48) in mouse results in early embryonic lethality
Biochemical and Biophysical Research Communications 354 (2007) 459–465 www.elsevier.com/locate/ybbrc
Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality J.M.M. Mu¨ller
a,1
, K. Deinhardt
a,2
, I. Rosewell b, G. Warren c, D.T. Shima
a,*
a
c
Endothelial Cell Biology Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK b Transgenic Services, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8002, USA Received 21 December 2006 Available online 8 January 2007
Abstract The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/ mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development. Ó 2007 Elsevier Inc. All rights reserved. Keywords: p97; CDC48; VCP; AAA ATPase; Mouse; Knock-out; Early embryonic lethality; Cell cycle; Embryogenesis; Ubiquitin; Folding
p97 is a member of the AAA family of ATPases (ATPases associated with diverse cellular activities), which includes metalloproteases, proteins involved in vesicle and organelle biogenesis, cell-cycle regulators, transcription factors and components of the 26 S proteasome. As a general mechanism, they couple ATP hydrolysis to unfolding of proteins or to dissociating proteins from large cellular structures [1–3]. p97 is a highly conserved protein, with homologues identified in organisms ranging from archaebacteria to man [4–8]. Its expression underlies a complex regulation in mouse, and correlates with disease progression of solid tumors in humans [9,10]. Gene disruption has revealed that p97 is an essential gene in Saccromy-
*
Corresponding author. Present address: R&D Consultants, 75 Lancaster Ave, Barnet, Herts EN4 0ES, UK. E-mail address: [email protected] (D.T. Shima). 1 Present address: Oncology Research, Merck KGaA Germany, Frankfurterstr. 250, 64293 Darmstadt, Germany. 2 Present address: Molecular NeuroPathoBiology Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.206
ces cerevisiae, Drosophila melanogaster and Trypanosoma brucei suggesting that it plays a fundamental role within both unicellular and multicellular organisms [11–13]. Data from diverse origins implicate p97 in a variety of cellular processes, such as cell cycle progression, apoptosis, protein degradation and organelle biogenesis, and it has been suggested that specific adaptor proteins act to regulate and target p97 activity into various pathways within the cell [14]. Genetic analysis identified CDC48 as a cell division cycle (CDC) gene and demonstrated that p97 is essential for normal progression through mitosis, most likely by regulating microtubule dynamics [5,15]. p97 is also implicated in a pathway that prevents apoptosis. It is a target gene for Pim-1, a protein involved in the gp130-STAT3 signalling pathway, which promotes cell cycle progression and prevents apoptosis [16]. In agreement with this finding, RNAi-based inhibition of p97 in HeLa cells reduces cell proliferation and promotes apoptosis [17]. p97 plays a role in ER dislocation and ubiquitin-proteasome dependent proteolysis. It interacts with polypeptide substrates at the ER membrane and releases them into
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the cytosol (Ye Y, 2001). Furthermore, p97 recognises ubiquitinated proteins and presents them to the 26 S proteasome [18–21]. Accordingly, recent studies revealed that p97 might play a role in inclusion body disorders, including muscle and neurodegenerative diseases [22–24]. Lastly, p97 is involved in organelle biogenesis. It plays a role in the formation of the endoplasmic reticulum, the nuclear envelope and Golgi cisternae [25–27]. The available biochemical and genetical data leave little doubt about a role of p97 as an essential cellular component, and its implication into pathological conditions in humans. However, experiments have not been extended to mammalian development to date. To better understand the physiological role of the p97 ATPase, we generated a p97 null allele in murine ES cells and mice. In this report, we show that homozygous null mice are not viable and die early during mammalian development. Materials and methods All reagents were obtained from BDH, Sigma or New England Biolabs unless indicated otherwise. ES cells and mouse strains were provided by Cancer Research UK Transgenic Services. Construction of the targeting vector. A vector containing a neomycin/ G418 resistance (neo+) gene and a thymidine kinase (tk) gene was used for gene targeting. The neo/tk-selection cassette was flanked by loxP-sites. Two adjacent BamHI fragments of the murine p97 gene [9] were cloned on either side of the floxed cassette. A 2.2 kb fragment (representing parts of the p97 promoter region) was inserted as 5 0 -fragment and a 4.2 kb fragment (corresponding to the basal promoter, the transcription start site, exon 1 and parts of intron 1 of p97) was inserted as 3 0 -fragment. Additionally, a third loxP-site [34] was inserted into the NsiI site of intron 1 to allow removal of the transcriptional unit of the p97 gene following Crespecific recombination (Fig. 1A). Targeting of p97 in embryonic stem (ES) cells. The 16.4 kb targeting vector was linearised with NotI and electroporated into 129P2/Ola ES cells. ES cells were positive selected 24 h post-transfection using 0.2 mg/ml G418 (Gibco BRL). Genomic DNA from individual G418-resistent clones was screened for homologous recombination and confirmed by Southern blotting (Fig. 1B–D). Cre-mediated removal of the floxed selection cassette was induced by transient transfection of a Cre recombinase expressing construct. ES cells were negative selected in 2 lM ganciclovir and clones were screened by Southern blotting and confirmed by PCR analysis (Fig. 2B–D). Preparation and analysis of DNA from ES cells, mice and embryos. Genomic DNA was isolated using standard protocols. For Southern blot analysis, genomic DNA from ES cells was digested with EcoRI or HincII and analysed. A 5 0 -end probe (0.5 kb BamHI/XhoI fragment corresponding to an external 5 0 -fragment of the targeted region) and a 3 0 -end probe (bp 164–465 of the mouse p97 coding region) [35] were used to confirm 5 0 - and 3 0 -ends of the targeted region, respectively (Fig. 1A). A neo probe (0.7 kb NcoI fragment of pcDNA3.1) was used to confirm single integration of the targeting vector into the genome (Fig. 1A). For genotyping, isolated DNA was subjected to PCR analysis using the following oligonucleotides: intron1S: 5 0 -GAGGATCACTGCAAGTTCAAGG-3 0 , intron1 A: 5 0 -CAGTAAGAGACCCTAGCTTGCC-3 0 , 838S: 5 0 -AAG ATATGGTCTTGTATCCTGG-3 0 , 949S: 5 0 -CTGACTCAAAAGTC AC AGGTTG-3 0 , 85A: 5 0 -CCGGGGCTGGACTCGCTGAAGCGG-3 0 , loxS: 5 0 -CTTCGTATAATGTATGCTATACG-3 0 (Fig. 2). Blastocysts were obtained by superovulation of 3- to 4-week old females and flushing the uterine horns at day 3 or 4 of pregnancy. They were genotyped by PCR followed by Southern blot analysis. PCR products were separated on agarose gel, blotted and hybridised with radiolabelled PCR product.
Generation of targeted mice. Mice were generated using standard procedures. Briefly, C57BL/6 blastocysts were injected with floxed ES cells (p97+/2lox) and implanted into pseudopregnant B6CBAF2 foster mothers. Germ line-transmitting chimeric males were backcrossed to C57BL/6 females to generate p97+/2lox mice. Heterozygous p97+/ mice were generated by crossing p97+/2lox mice to ZP3-Cre mice, which express Cre recombinase in the oocytes prior to the first cell division [29]. Homozygous p97/ and p972lox/2lox mice were generated by intercrossing p97+/ or p97+/2lox mice, respectively.
Results and discussion Targeting of the p97 gene Mus musculus contains two distinct genes for p97, one representing a non-functional pseudogene [9]. The coding region of the functional gene is interrupted by 16 introns and encompasses 20.4 kb. To generate a conditional (floxed) knockout allele of p97, we inserted a loxP-flanked neo/tk selectable marker upstream of the basal p97 promoter and a third loxP site into intron 1. Accordingly, loxP sites flanked 3kb of the p97 gene comprising the basal promoter, the transcription start site, exon 1 (encoding the 5 0 untranslated region, ATG start codon, and six amino acids) and most of intron 1 (Fig. 1A). Following introduction of the targeting vector into 129P2/Ola ES cells, integration of the selection cassette via homologous recombination was verified by Southern blot analyses using 5 0 - and 3 0 -specific probes. As seen in Fig. 1B and C, integration of the selection cassette increased the size of EcoRI and HincII fragments at the 5 0 - and 3 0 -end, respectively. We used a neo-specific probe to confirm single integration of the neo cassette into the genome (Fig. 1D). Furthermore, co-integration of the third loxP site into intron 1 was verified by PCR analysis. In these experiments homologous recombination occurred with 6% efficiency, however only 1% of the targeted cells had co-integrated the third loxP site into the gene. The resulting p97 allele was designated p973lox (Fig. 1A). We next removed the selection cassette in ES cells through Cre-mediated recombination (Fig. 2A) and screened the cells by Southern blot analysis. As seen in Fig. 2B, Cre-mediated recombination decreased the size of an EcoRI fragment at the 5 0 -end of the gene. Cells with conditional null genotypes, p97+/2lox, were further examined by two independent PCR analyses to confirm the second and the third loxP site (Fig. 2C and D; see also Fig. 2A for primer annealing sites). Generation of heterozygous mutant mice Based on the results obtained in lower organisms, we expected that p97 deficiency would result in lethality. Therefore, we decided to generate mice with a conditional null allele, which permits gene-inactivation at any stage of embryonic and postnatal life and in any tissue [28]. Mice were derived from p97+/2lox ES cells in a C57BL/6 background. We used PCR analysis to show that p97+/2lox mice
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Fig. 1. Targeting of the p97gene. (A) A map of the targeting vector and the configuration of the p97 gene surrounding the targeted region before and after homologous recombination. Gene targeting altered the restriction pattern of the locus. An EcoRI fragment at the 5 0 -end of the gene increased from 5.5 to 6.2 kb and was detected by Southern blotting using a 5 0 -end probe. A HincII fragment at the 3 0 -end increased from 7.5 to 9.0 kb and was detected using a 3 0 -end probe. The integrated selection cassette was detected as 4.0 kb fragment with a neo-specific probe. Exons are indicated by grey boxes and loxP-sites by black triangles. Restriction sites are marked for EcoRI (E), BamHI (B), XhoI (X) and HincII (H). (B–D) Identification of p97+/3lox ES cell clones. (B) Genomic DNA of the ES cell clones with different genotypes was digested with EcoRI and analysed by Southern blotting using a 5 0 -end probe. Targeted clones give two bands of 5.5 and 6.2 kb for wild-type and 3lox alleles, respectively. (C) Positive clones were further characterised by using a 3 0 -end probe to identify wild-type and 3lox alleles as HincII fragments of 7.5 and 9.0 kb, respectively. (D) The integrated neo gene was detected with a neo-specific probe as EcoRI fragment of 4.0 kb.
transmitted the floxed 2lox allele to their offspring. For this, a primer pair that covered the third loxP site was used to amplify wild-type and 2lox alleles as two distinguishable fragments (Fig. 2E, see also Fig. 2A). To generate mice with a p97 null allele we bred p97+/2lox mice with ZP3Cre mice that expressed Cre recombinase in growing oocytes prior to completion of the first meiotic division, thus giving rise to recombined alleles in all tissues [29]. We confirmed deletion of the 3 kb-floxed p97 segment in the resulting offspring with PCR analysis by using a primer pair that generated a PCR fragment only in case of p97 null alleles (Fig. 2F, see also Fig. 2A). Cre-mediated recombination occurred in 71% of the offspring, and p97+/ mice were born at normal Mendelian ratio. Macroscopic exam-
ination of p97+/ and p97+/2lox embryos and born mice suggested that the heterozygous mutant mice developed normally. Adult p97+/ and p97+/2lox mice were of normal size and appeared to have normal fertility. Analysis of p97 RNA (data not shown) and protein levels in ES cells or different organs from adult mice revealed that p97 was expressed at similar levels in the three different genotypes (Fig. 3A). In addition, p97 protein levels were similar in organs derived from p97+/+ and p97+/2lox littermates at developmental stage E16.5 (Fig. 3B). We also analysed total cell lysate from wild-type and heterozygous p97+/ and p97+/2lox ES cells using an antip97 antibody to confirm that no truncated p97 protein was expressed from the null or 2lox alleles (Fig. 4).
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Fig. 2. Generation of heterozygous targeted mice. (A) Schematic representation of the p97+ allele, the resulting p973lox allele after gene targeting, and the p972lox and p97- alleles after Cre expression. Exons are indicated by grey boxes and loxP-sites by black triangles. The transcription start site and primer annealing sites for PCR analysis are shown. (B–D) p97+/3lox ES cell clones were transfected with Cre recombinase and analysed. (B) Genomic DNA was digested with EcoRI and analysed by Southern blotting using the 5 0 -end probe. A 6.2 kb fragment of the 3lox allele decreased to 5.5 kb following recombination. (C, D) PCR analysis was carried out to verify the second loxP site using primer pair loxS/85A (C) and the third loxP-site using primer pair intron1S/intron1 A (D). (E,F) Heterozygous p97+/2lox mice were crossed to ZP3-Cre expressing mice to generate mice with ZP3-Cre p97+/2lox genotype. Mice were intercrossed to generate ZP3-Cre p97+/ mice and they were analysed by PCR analysis. (E) Primer pair intron1S/intron1A was used to detect deletion of the third loxP site in p97+/ mice. (F) Primer pair -838 S/intron1 A was used to detect recombination of the 3 kb-floxed p97 segment containing the promoter region, the transcription start site, exon 1 and part of intron 1 (see A).
Generation of homozygous mutant mice Next, we aimed to generate homozygous p97/ mice. For this, heterozygous p97+/ mice were bred and weaned pups were genotyped by PCR analysis. Again, wild-type p97+/+ and heterozygous p97+/ mice were born at normal Mendelian ratio. In contrast, no homozygous p97/ null mice were detected among born neonates indicating that p97 is an essential gene in mouse (Table 1). We then backcrossed p97+/ mice into outbred CD-1 background to examine whether changing the genetic background of the
mice might influence the mutant phenotype. We chose CD-1 as genetic background as it has been described as having less severe mutant phenotypes than inbred strains [30]. However, genotyping identified no homozygous p97/ mice either in mixed genetic background or after 5 generations of CD-1 backcrossing. The role of p97 during early embryonic development In order to determine how early lethality occurred during development, we analysed E3.5 embryos at blastocyst
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Fig. 3. p97 protein levels in ES cells and mice. Protein was extracted from ES cells and mouse organs of different genotypic origins and analysed by Western blotting using anti-p97 and anti-actin antibodies. (A) Analysis of p97 protein levels in kidney and brain from adult littermates and ES cells with p97+/+, p97+/2lox and p97+/ genotypes. (B) Analysis of p97 protein levels in different organs from E16.5 p97+/+ and p97+/2lox littermates.
Fig. 4. Western blot analysis of p97+/+, p97+/2lox and p97+/ ES cells. Protein was extracted from ES cells with different genotypes and analysed by Western blotting using an anti-p97 antibody. No truncated protein could be detected. MW, molecular weight, given in thousands.
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heterozygous p97+/2lox intercrosses at developmental stages ranging from E4.5 to live-born mice (Table 2). Furthermore, no p972lox/ mice were detected from p97+/2lox x p97+/ crosses (data not shown). These data suggest that p97 expression from p972lox alleles was severely impaired and corroborate the finding that p97 deficiency results in early embryonic lethality. The result that null and floxed alleles behaved similar suggests that insertion of one of the two loxP sites interfered with p97 gene expression. According to a database search using the Biobase Transfac database (release 6.0) and Genomatix GEMS (Genome Exploring and Modeling Software) no known regulatory sequences were interrupted by integration of either loxP site into the p97 locus. However, regulatory elements within the first intron are still poorly characterised, and interference of the 3 0 -intronic loxP site cannot be entirely excluded. To date, p97 has been implicated in a variety of seemingly unrelated cellular events, such as cell cycle progression, apoptosis, ER dislocation, ubiquitin-proteasome proteolysis and membrane fusion. Most of these cellular processes are involved in or modulated during cell proliferation and thus imply an essential function of p97 in this process. A role for p97 in cell cycle progression has been documented extensively in a variety of different organisms. In S. cerevisiae, CDC48 was first isolated as a cell division cycle gene and certain cdc48 mutants arrest at G2/M with large budded cells, undivided nuclei, and long microtubules, which spread aberrantly from an unseparated spindle pole body into the cytoplasm [11]. These observations were elucidated further by the evidence that p97 modulates microtubule dynamics at the end of mitosis in Xenopus [15]. Moreover, p97’s crucial function in cell cycle progression is supported by overexpression studies of dominant-negative forms of p97 in T. brucei [12,17]. p97 has been involved in ubiquitin-proteasome proteolysis and its function in proteolysis can also be linked to cell proliferation. RNA interference of p97 reduces proliferation of HeLa cells and causes the accumulation of polyubiquitinated proteins [17]. Furthermore, p97 is implicated in the degradation of proteins involved in cell cycle progression, such as cyclin B, cyclin E and the G1-CDK inhibitor Far1P [18–20]. Lastly, p97 is required for organelle biogenesis, and interestingly, homotypic membrane fusion events of organelles are highly coordinated with cell proliferation. For example,
Table 1 Genotypes of mice and embryos from p97+/ intercrossings Age
Live-born mice E3.5 (blastocysts)
No. of offsprings
106 9
P97 genotype p97+/+
p97+/
p97/
32 2
74 7
0 0
stage. No null mutants were identified by this approach indicating that p97 deficiency results in early embryonic lethality (Table 1). To our surprise, we also obtained no homozygous p972lox/2lox mutants when analysing offspring from
Table 2 Genotypes of mice and embryos from p97+/2lox intercrossings Age
Live-born mice E16.5 E15.5 E10.5 E9.5 E4.5 (blastocysts)
No. of offsprings
94 9 16 35 12 9
p97 genotype p97+/+
p97+/2lox
p972lox/2lox
31 7 3 16 2 4
63 2 13 19 10 5
0 0 0 0 0 0
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p97 promotes the biogenesis of several different organelles in cell-free assays that replicate post-mitotic fusion events, including the Golgi apparatus, the endoplasmic reticulum and the nuclear envelope [25,26,31]. Interestingly, these fusion events also involve the recognition of ubiquitinated substrates, suggesting that ubiquitin recognition is a common feature of p97-mediated reactions [32,33]. In conclusion, our finding that p97 gene ablation in mouse leads to early embryonic lethality suggests that p97 coordination of cellular events with cell proliferation and survival is an essential feature of mammalian development. Acknowledgments We thank Hemmo Meyer for the p97 antibody, Mike Mitchell for the database searches and Christiana Ruhrberg for critical reading of the manuscript. This work was supported by grants from the Boehringer Ingelheim Fonds (J.M.M.), Cancer Research UK and Burroughs Wellcome Funds (D.T.S.). References [1] A.N. Lupas, J. Martin, AAA proteins, Curr. Opin. Struct. Biol. 12 (2002) 746–753. [2] F. Confalonieri, M. Duguet, A 200-amino acid ATPase module in search of a basic function, Bioessays 17 (1995) 639–650. [3] S. Patel, M. Latterich, The AAA team: related ATPases with diverse functions, Trends Cell Biol. 8 (1998) 65–71. [4] F. Confalonieri, J. Marsault, M. Duguet, SAV, an archaebacterial gene with extensive homology to a family of highly conserved eukaryotic ATPases, J. Mol. Biol. 235 (1994) 396–401. [5] D. Moir, S.E. Stewart, B.C. Osmond, D. Botstein, Cold-sensitive celldivision-cycle mutants of yeast: isolation, properties, and pseudoreversion studies, Genetics 100 (1982) 547–563. [6] M. Pinter, G. Jekely, R.J. Szepesi, A. Farkas, U. Theopold, H.E. Meyer, D. Lindholm, D.R. Nassel, D. Hultmark, P. Friedrich, TER94, a Drosophila homolog of the membrane fusion protein CDC48/p97, is accumulated in nonproliferating cells: in the reproductive organs and in the brain of the imago, Insect Biochem. Mol. Biol. 28 (1998) 91–98. [7] H.S. Feiler, T. Desprez, V. Santoni, J. Kronenberger, M. Caboche, J. Traas, The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells, EMBO J. 14 (1995) 5626–5637. [8] J.M. Peters, M.J. Walsh, W.W. Franke, An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF, EMBO J. 9 (1990) 1757–1767. [9] J.M. Muller, H.H. Meyer, C. Ruhrberg, G.W. Stamp, G. Warren, D.T. Shima, The mouse p97 (CDC48) gene. Genomic structure, definition of transcriptional regulatory sequences, gene expression, and characterization of a pseudogene, J. Biol. Chem. 274 (1999) 10154–10162. [10] S. Yamamoto, Y. Tomita, Y. Hoshida, M. Sakon, M. Kameyama, S. Imaoka, M. Sekimoto, S. Nakamori, M. Monden, K. Aozasa, Expression of valosin-containing protein in colorectal carcinomas as a predictor for disease recurrence and prognosis, Clin. Cancer Res. 10 (2004) 651–657. [11] K.U. Frohlich, H.W. Fries, M. Rudiger, R. Erdmann, D. Botstein, D. Mecke, Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression, J. Cell Biol. 114 (1991) 443–453.
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Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality J.M.M. Mu¨ller
a,1
, K. Deinhardt
a,2
, I. Rosewell b, G. Warren c, D.T. Shima
a,*
a
c
Endothelial Cell Biology Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK b Transgenic Services, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8002, USA Received 21 December 2006 Available online 8 January 2007
Abstract The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/ mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development. Ó 2007 Elsevier Inc. All rights reserved. Keywords: p97; CDC48; VCP; AAA ATPase; Mouse; Knock-out; Early embryonic lethality; Cell cycle; Embryogenesis; Ubiquitin; Folding
p97 is a member of the AAA family of ATPases (ATPases associated with diverse cellular activities), which includes metalloproteases, proteins involved in vesicle and organelle biogenesis, cell-cycle regulators, transcription factors and components of the 26 S proteasome. As a general mechanism, they couple ATP hydrolysis to unfolding of proteins or to dissociating proteins from large cellular structures [1–3]. p97 is a highly conserved protein, with homologues identified in organisms ranging from archaebacteria to man [4–8]. Its expression underlies a complex regulation in mouse, and correlates with disease progression of solid tumors in humans [9,10]. Gene disruption has revealed that p97 is an essential gene in Saccromy-
*
Corresponding author. Present address: R&D Consultants, 75 Lancaster Ave, Barnet, Herts EN4 0ES, UK. E-mail address: [email protected] (D.T. Shima). 1 Present address: Oncology Research, Merck KGaA Germany, Frankfurterstr. 250, 64293 Darmstadt, Germany. 2 Present address: Molecular NeuroPathoBiology Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.206
ces cerevisiae, Drosophila melanogaster and Trypanosoma brucei suggesting that it plays a fundamental role within both unicellular and multicellular organisms [11–13]. Data from diverse origins implicate p97 in a variety of cellular processes, such as cell cycle progression, apoptosis, protein degradation and organelle biogenesis, and it has been suggested that specific adaptor proteins act to regulate and target p97 activity into various pathways within the cell [14]. Genetic analysis identified CDC48 as a cell division cycle (CDC) gene and demonstrated that p97 is essential for normal progression through mitosis, most likely by regulating microtubule dynamics [5,15]. p97 is also implicated in a pathway that prevents apoptosis. It is a target gene for Pim-1, a protein involved in the gp130-STAT3 signalling pathway, which promotes cell cycle progression and prevents apoptosis [16]. In agreement with this finding, RNAi-based inhibition of p97 in HeLa cells reduces cell proliferation and promotes apoptosis [17]. p97 plays a role in ER dislocation and ubiquitin-proteasome dependent proteolysis. It interacts with polypeptide substrates at the ER membrane and releases them into
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the cytosol (Ye Y, 2001). Furthermore, p97 recognises ubiquitinated proteins and presents them to the 26 S proteasome [18–21]. Accordingly, recent studies revealed that p97 might play a role in inclusion body disorders, including muscle and neurodegenerative diseases [22–24]. Lastly, p97 is involved in organelle biogenesis. It plays a role in the formation of the endoplasmic reticulum, the nuclear envelope and Golgi cisternae [25–27]. The available biochemical and genetical data leave little doubt about a role of p97 as an essential cellular component, and its implication into pathological conditions in humans. However, experiments have not been extended to mammalian development to date. To better understand the physiological role of the p97 ATPase, we generated a p97 null allele in murine ES cells and mice. In this report, we show that homozygous null mice are not viable and die early during mammalian development. Materials and methods All reagents were obtained from BDH, Sigma or New England Biolabs unless indicated otherwise. ES cells and mouse strains were provided by Cancer Research UK Transgenic Services. Construction of the targeting vector. A vector containing a neomycin/ G418 resistance (neo+) gene and a thymidine kinase (tk) gene was used for gene targeting. The neo/tk-selection cassette was flanked by loxP-sites. Two adjacent BamHI fragments of the murine p97 gene [9] were cloned on either side of the floxed cassette. A 2.2 kb fragment (representing parts of the p97 promoter region) was inserted as 5 0 -fragment and a 4.2 kb fragment (corresponding to the basal promoter, the transcription start site, exon 1 and parts of intron 1 of p97) was inserted as 3 0 -fragment. Additionally, a third loxP-site [34] was inserted into the NsiI site of intron 1 to allow removal of the transcriptional unit of the p97 gene following Crespecific recombination (Fig. 1A). Targeting of p97 in embryonic stem (ES) cells. The 16.4 kb targeting vector was linearised with NotI and electroporated into 129P2/Ola ES cells. ES cells were positive selected 24 h post-transfection using 0.2 mg/ml G418 (Gibco BRL). Genomic DNA from individual G418-resistent clones was screened for homologous recombination and confirmed by Southern blotting (Fig. 1B–D). Cre-mediated removal of the floxed selection cassette was induced by transient transfection of a Cre recombinase expressing construct. ES cells were negative selected in 2 lM ganciclovir and clones were screened by Southern blotting and confirmed by PCR analysis (Fig. 2B–D). Preparation and analysis of DNA from ES cells, mice and embryos. Genomic DNA was isolated using standard protocols. For Southern blot analysis, genomic DNA from ES cells was digested with EcoRI or HincII and analysed. A 5 0 -end probe (0.5 kb BamHI/XhoI fragment corresponding to an external 5 0 -fragment of the targeted region) and a 3 0 -end probe (bp 164–465 of the mouse p97 coding region) [35] were used to confirm 5 0 - and 3 0 -ends of the targeted region, respectively (Fig. 1A). A neo probe (0.7 kb NcoI fragment of pcDNA3.1) was used to confirm single integration of the targeting vector into the genome (Fig. 1A). For genotyping, isolated DNA was subjected to PCR analysis using the following oligonucleotides: intron1S: 5 0 -GAGGATCACTGCAAGTTCAAGG-3 0 , intron1 A: 5 0 -CAGTAAGAGACCCTAGCTTGCC-3 0 , 838S: 5 0 -AAG ATATGGTCTTGTATCCTGG-3 0 , 949S: 5 0 -CTGACTCAAAAGTC AC AGGTTG-3 0 , 85A: 5 0 -CCGGGGCTGGACTCGCTGAAGCGG-3 0 , loxS: 5 0 -CTTCGTATAATGTATGCTATACG-3 0 (Fig. 2). Blastocysts were obtained by superovulation of 3- to 4-week old females and flushing the uterine horns at day 3 or 4 of pregnancy. They were genotyped by PCR followed by Southern blot analysis. PCR products were separated on agarose gel, blotted and hybridised with radiolabelled PCR product.
Generation of targeted mice. Mice were generated using standard procedures. Briefly, C57BL/6 blastocysts were injected with floxed ES cells (p97+/2lox) and implanted into pseudopregnant B6CBAF2 foster mothers. Germ line-transmitting chimeric males were backcrossed to C57BL/6 females to generate p97+/2lox mice. Heterozygous p97+/ mice were generated by crossing p97+/2lox mice to ZP3-Cre mice, which express Cre recombinase in the oocytes prior to the first cell division [29]. Homozygous p97/ and p972lox/2lox mice were generated by intercrossing p97+/ or p97+/2lox mice, respectively.
Results and discussion Targeting of the p97 gene Mus musculus contains two distinct genes for p97, one representing a non-functional pseudogene [9]. The coding region of the functional gene is interrupted by 16 introns and encompasses 20.4 kb. To generate a conditional (floxed) knockout allele of p97, we inserted a loxP-flanked neo/tk selectable marker upstream of the basal p97 promoter and a third loxP site into intron 1. Accordingly, loxP sites flanked 3kb of the p97 gene comprising the basal promoter, the transcription start site, exon 1 (encoding the 5 0 untranslated region, ATG start codon, and six amino acids) and most of intron 1 (Fig. 1A). Following introduction of the targeting vector into 129P2/Ola ES cells, integration of the selection cassette via homologous recombination was verified by Southern blot analyses using 5 0 - and 3 0 -specific probes. As seen in Fig. 1B and C, integration of the selection cassette increased the size of EcoRI and HincII fragments at the 5 0 - and 3 0 -end, respectively. We used a neo-specific probe to confirm single integration of the neo cassette into the genome (Fig. 1D). Furthermore, co-integration of the third loxP site into intron 1 was verified by PCR analysis. In these experiments homologous recombination occurred with 6% efficiency, however only 1% of the targeted cells had co-integrated the third loxP site into the gene. The resulting p97 allele was designated p973lox (Fig. 1A). We next removed the selection cassette in ES cells through Cre-mediated recombination (Fig. 2A) and screened the cells by Southern blot analysis. As seen in Fig. 2B, Cre-mediated recombination decreased the size of an EcoRI fragment at the 5 0 -end of the gene. Cells with conditional null genotypes, p97+/2lox, were further examined by two independent PCR analyses to confirm the second and the third loxP site (Fig. 2C and D; see also Fig. 2A for primer annealing sites). Generation of heterozygous mutant mice Based on the results obtained in lower organisms, we expected that p97 deficiency would result in lethality. Therefore, we decided to generate mice with a conditional null allele, which permits gene-inactivation at any stage of embryonic and postnatal life and in any tissue [28]. Mice were derived from p97+/2lox ES cells in a C57BL/6 background. We used PCR analysis to show that p97+/2lox mice
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Fig. 1. Targeting of the p97gene. (A) A map of the targeting vector and the configuration of the p97 gene surrounding the targeted region before and after homologous recombination. Gene targeting altered the restriction pattern of the locus. An EcoRI fragment at the 5 0 -end of the gene increased from 5.5 to 6.2 kb and was detected by Southern blotting using a 5 0 -end probe. A HincII fragment at the 3 0 -end increased from 7.5 to 9.0 kb and was detected using a 3 0 -end probe. The integrated selection cassette was detected as 4.0 kb fragment with a neo-specific probe. Exons are indicated by grey boxes and loxP-sites by black triangles. Restriction sites are marked for EcoRI (E), BamHI (B), XhoI (X) and HincII (H). (B–D) Identification of p97+/3lox ES cell clones. (B) Genomic DNA of the ES cell clones with different genotypes was digested with EcoRI and analysed by Southern blotting using a 5 0 -end probe. Targeted clones give two bands of 5.5 and 6.2 kb for wild-type and 3lox alleles, respectively. (C) Positive clones were further characterised by using a 3 0 -end probe to identify wild-type and 3lox alleles as HincII fragments of 7.5 and 9.0 kb, respectively. (D) The integrated neo gene was detected with a neo-specific probe as EcoRI fragment of 4.0 kb.
transmitted the floxed 2lox allele to their offspring. For this, a primer pair that covered the third loxP site was used to amplify wild-type and 2lox alleles as two distinguishable fragments (Fig. 2E, see also Fig. 2A). To generate mice with a p97 null allele we bred p97+/2lox mice with ZP3Cre mice that expressed Cre recombinase in growing oocytes prior to completion of the first meiotic division, thus giving rise to recombined alleles in all tissues [29]. We confirmed deletion of the 3 kb-floxed p97 segment in the resulting offspring with PCR analysis by using a primer pair that generated a PCR fragment only in case of p97 null alleles (Fig. 2F, see also Fig. 2A). Cre-mediated recombination occurred in 71% of the offspring, and p97+/ mice were born at normal Mendelian ratio. Macroscopic exam-
ination of p97+/ and p97+/2lox embryos and born mice suggested that the heterozygous mutant mice developed normally. Adult p97+/ and p97+/2lox mice were of normal size and appeared to have normal fertility. Analysis of p97 RNA (data not shown) and protein levels in ES cells or different organs from adult mice revealed that p97 was expressed at similar levels in the three different genotypes (Fig. 3A). In addition, p97 protein levels were similar in organs derived from p97+/+ and p97+/2lox littermates at developmental stage E16.5 (Fig. 3B). We also analysed total cell lysate from wild-type and heterozygous p97+/ and p97+/2lox ES cells using an antip97 antibody to confirm that no truncated p97 protein was expressed from the null or 2lox alleles (Fig. 4).
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Fig. 2. Generation of heterozygous targeted mice. (A) Schematic representation of the p97+ allele, the resulting p973lox allele after gene targeting, and the p972lox and p97- alleles after Cre expression. Exons are indicated by grey boxes and loxP-sites by black triangles. The transcription start site and primer annealing sites for PCR analysis are shown. (B–D) p97+/3lox ES cell clones were transfected with Cre recombinase and analysed. (B) Genomic DNA was digested with EcoRI and analysed by Southern blotting using the 5 0 -end probe. A 6.2 kb fragment of the 3lox allele decreased to 5.5 kb following recombination. (C, D) PCR analysis was carried out to verify the second loxP site using primer pair loxS/85A (C) and the third loxP-site using primer pair intron1S/intron1 A (D). (E,F) Heterozygous p97+/2lox mice were crossed to ZP3-Cre expressing mice to generate mice with ZP3-Cre p97+/2lox genotype. Mice were intercrossed to generate ZP3-Cre p97+/ mice and they were analysed by PCR analysis. (E) Primer pair intron1S/intron1A was used to detect deletion of the third loxP site in p97+/ mice. (F) Primer pair -838 S/intron1 A was used to detect recombination of the 3 kb-floxed p97 segment containing the promoter region, the transcription start site, exon 1 and part of intron 1 (see A).
Generation of homozygous mutant mice Next, we aimed to generate homozygous p97/ mice. For this, heterozygous p97+/ mice were bred and weaned pups were genotyped by PCR analysis. Again, wild-type p97+/+ and heterozygous p97+/ mice were born at normal Mendelian ratio. In contrast, no homozygous p97/ null mice were detected among born neonates indicating that p97 is an essential gene in mouse (Table 1). We then backcrossed p97+/ mice into outbred CD-1 background to examine whether changing the genetic background of the
mice might influence the mutant phenotype. We chose CD-1 as genetic background as it has been described as having less severe mutant phenotypes than inbred strains [30]. However, genotyping identified no homozygous p97/ mice either in mixed genetic background or after 5 generations of CD-1 backcrossing. The role of p97 during early embryonic development In order to determine how early lethality occurred during development, we analysed E3.5 embryos at blastocyst
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Fig. 3. p97 protein levels in ES cells and mice. Protein was extracted from ES cells and mouse organs of different genotypic origins and analysed by Western blotting using anti-p97 and anti-actin antibodies. (A) Analysis of p97 protein levels in kidney and brain from adult littermates and ES cells with p97+/+, p97+/2lox and p97+/ genotypes. (B) Analysis of p97 protein levels in different organs from E16.5 p97+/+ and p97+/2lox littermates.
Fig. 4. Western blot analysis of p97+/+, p97+/2lox and p97+/ ES cells. Protein was extracted from ES cells with different genotypes and analysed by Western blotting using an anti-p97 antibody. No truncated protein could be detected. MW, molecular weight, given in thousands.
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heterozygous p97+/2lox intercrosses at developmental stages ranging from E4.5 to live-born mice (Table 2). Furthermore, no p972lox/ mice were detected from p97+/2lox x p97+/ crosses (data not shown). These data suggest that p97 expression from p972lox alleles was severely impaired and corroborate the finding that p97 deficiency results in early embryonic lethality. The result that null and floxed alleles behaved similar suggests that insertion of one of the two loxP sites interfered with p97 gene expression. According to a database search using the Biobase Transfac database (release 6.0) and Genomatix GEMS (Genome Exploring and Modeling Software) no known regulatory sequences were interrupted by integration of either loxP site into the p97 locus. However, regulatory elements within the first intron are still poorly characterised, and interference of the 3 0 -intronic loxP site cannot be entirely excluded. To date, p97 has been implicated in a variety of seemingly unrelated cellular events, such as cell cycle progression, apoptosis, ER dislocation, ubiquitin-proteasome proteolysis and membrane fusion. Most of these cellular processes are involved in or modulated during cell proliferation and thus imply an essential function of p97 in this process. A role for p97 in cell cycle progression has been documented extensively in a variety of different organisms. In S. cerevisiae, CDC48 was first isolated as a cell division cycle gene and certain cdc48 mutants arrest at G2/M with large budded cells, undivided nuclei, and long microtubules, which spread aberrantly from an unseparated spindle pole body into the cytoplasm [11]. These observations were elucidated further by the evidence that p97 modulates microtubule dynamics at the end of mitosis in Xenopus [15]. Moreover, p97’s crucial function in cell cycle progression is supported by overexpression studies of dominant-negative forms of p97 in T. brucei [12,17]. p97 has been involved in ubiquitin-proteasome proteolysis and its function in proteolysis can also be linked to cell proliferation. RNA interference of p97 reduces proliferation of HeLa cells and causes the accumulation of polyubiquitinated proteins [17]. Furthermore, p97 is implicated in the degradation of proteins involved in cell cycle progression, such as cyclin B, cyclin E and the G1-CDK inhibitor Far1P [18–20]. Lastly, p97 is required for organelle biogenesis, and interestingly, homotypic membrane fusion events of organelles are highly coordinated with cell proliferation. For example,
Table 1 Genotypes of mice and embryos from p97+/ intercrossings Age
Live-born mice E3.5 (blastocysts)
No. of offsprings
106 9
P97 genotype p97+/+
p97+/
p97/
32 2
74 7
0 0
stage. No null mutants were identified by this approach indicating that p97 deficiency results in early embryonic lethality (Table 1). To our surprise, we also obtained no homozygous p972lox/2lox mutants when analysing offspring from
Table 2 Genotypes of mice and embryos from p97+/2lox intercrossings Age
Live-born mice E16.5 E15.5 E10.5 E9.5 E4.5 (blastocysts)
No. of offsprings
94 9 16 35 12 9
p97 genotype p97+/+
p97+/2lox
p972lox/2lox
31 7 3 16 2 4
63 2 13 19 10 5
0 0 0 0 0 0
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