Mol. Hum. Reprod. Advance Access originally published online on February 26, 2008
Molecular Human Reproduction 2008 14(4):207-213; doi:10.1093/molehr/gan009
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Fas-associated factor (FAF1) is required for the early cleavage-stages of mouse embryo
1Institute of Human Genetics, Faculty of Medicine, University of Göttingen, 37073 Göttingen, Germany 2Institute of Biochemistry, Faculty of Medicine, University of Göttingen, 37073 Göttingen, Germany 3Department of Molecular Cell Biology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany 4Department of Anatomy and Cell Biology, Faculty of Medicine, University of Giessen, 35385 Giessen, Germany
5 Correspondence address. Tel: +49-551-397522; Fax: +49-551-399303; E-mail: iadham{at}gwdg.de
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Abstract |
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FAF1 was initially isolated as a Fas-associated factor and was subsequently found to interact with a subset of additional proteins that are involved in many cellular events including Fas-mediated apoptosis, heat shock signalling pathways and ubiquitin-dependent processes. Here, we describe that the 74-kDa FAF1 is ubiquitously expressed, while the expression of its post-translational-processed 49-kDa isoform is restricted to post-meiotic male germ cells. In ovary, FAF1 protein is localized predominantly in the cytoplasm of oocytes in all follicle stages. To determine the function of FAF1 in vivo, we analysed a mouse mutant line in which a gene trap vector was inserted in the Faf1 locus. The mutation disrupts the Faf1 and leads to lethality of the Faf1GT/GT embryos near the 2-cell stage. Analysis of FAF1 expression revealed that the protein is present in early preimplantation stages, while embryonic expression of Faf1 mRNA becomes appreciable at 4-cell stage. These results indicate that the death of Faf1GT/GT at the 2-cell stage may coincide with the depletion of maternal FAF1 in these embryos. Thus, our results indicate that the FAF1 gene product is necessary for early embryonic development.
Key words: FAF1/spermatogenesis/oogenesis/early preimplantation stages
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Introduction |
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The Fas-associated factor gene (Faf1) encodes a multidomain protein that mediates a variety of biological functions, which were postulated from the predicted protein structure and from specific interaction with putative partners. Specific binding of the N-terminal domain of FAF1 with Fas ligand, nuclear factor
B p65-subunit (NF-B) and heatshock protein 70 (HSP70) has been determined. The interaction of FAF1 with these proteins has been found to initiate Fas-mediated apoptosis, inhibition of NF-B and HSP70-chaperone activity (Chu et al., 1995; Ryu et al., 2003; Park et al., 2004; Kim et al., 2005). Faf1 is evolutionarily conserved and ubiquitously expressed as a 2.8-kb mRNA that encodes a 74-kDa protein (Ryu et al., 1999). The FAF1 protein contains an internal ubiquitin-associated (UBA) and a C-terminal ubiquitin-like (UBX) domain. Both domains are well characterized in the mammalian protein NSFL1c/p47 and the UBX proteins of Saccharomyces cerevisiae (Buchberger et al., 2001; Dreveny et al., 2004; Schuberth et al., 2004). These proteins bind to ubiquitinated proteins and valosin-containing protein (VCP/p97) via the UBA and UBX domain, respectively (Meyer et al., 2002; McNeill et al., 2004; Schuberth et al., 2004; Song et al., 2005). Proteins containing UBA and UBX domains are believed to be adapter proteins in the complex that facilitate the degradation of polyubiquitinated proteins by the proteasome (Dai and Li, 2001).
The physiological function of FAF1, particularly its role in embryogenesis, has not been resolved. The goal of this study was to determine the consequence of FAF1 deficiency during mouse development. We analysed a mouse mutant line, in which a gene trap vector has been inserted in intron 7 of Faf1. The mutation disrupts the Faf1 locus and leads to lethality of the Faf1GT/GT embryos near the 2-cell stage. To determine the cause of early embryonic lethality, we have investigated the expression of Faf1 during gametogenesis and early embryonic development.
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Materials and Methods |
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Generation of the Faf1 gene trap mouse
The cassette of gene trap vector pMS1 consists of a splice acceptor (SA) from the engrailed 2 (En-2) gene followed by the encephalomyocarditis virus internal ribosome entry site (IRES) that directs the translation of a fusion protein with β-galactosidase and neomycin-resistance activities (βGeo), and ends with the simian virus 40 late poly (A) signal. The ES-cell line 2C98, containing an insertion of the gene trap vector (pMS1) within the Faf1 gene, was generated as described previously (Salminen et al., 1998). The 129/Sv-derived ES cell clone was used in morula aggregations according to published procedures to obtain chimeric male founders that were mated with outbred NMRI females. Heterozygous progeny was firstly genotyped by PCR using primers localized in βGeo cassette (Chawdhury et al., 1997). Heterozygous animals were then intercrossed to produce F2 animals.
All animal experimentations were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Göttingen.
Construction of genomic phage library
To clone the site of vector integration, a genomic phage library was generated in the 
DASH-II vector (Stratagene, La Jolla, USA) according to standard procedures. Two independent recombinant phage clones carrying inserts of 21- and 17.5-kb were isolated with βGeo probe. Regions flanking the integrated vector were sequenced and searched using Blast against mouse genome databases.
Genotyping of Faf1-trapped mice and preimplantation embryos
For genotyping Faf1-trapped mice by Southern blot analysis, genomic DNA extracted from tail biopsies was digested with BamHI, electrophoresed and blotted onto nitrocellulose membrane. The 0.6-kb genomic fragment located 3' of the gene trap integration site was radioactively labelled and used to probe the Southern blots. Hybridization was carried out at 65°C overnight in the following solution: 5x SSC/5x Denhardt's solution, 0.1% SDS and 100 µg/ml denatured salmon sperm DNA. Filters were washed twice at 65°C at a final stringency of 0.2x SSC/0.1% SDS. Primers to amplify the 0.6-kb probe were 5'-ACATCACTTTACCTGCTGAGC-3' and 5'-TCCCTGAAGCCTATTTAGAG-3'. Embryos and mice were genotyped by PCR using three primers. A single antisense primer, FR3: 5'-GCGTTGGGAAGACTACAGGA-3', was designed to amplify both wild-type and trapped loci. The first sense primer, FF4: 5'-CACGCTGGGACATACAAATG-3', was designed to amplify the wild-type locus. The second sense primer, MR3: 5'-GCGTTGGGAAGACTACAGGA-3', was designed to amplify the trapped allele. Thermal cycling was carried out for 35 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. PCR primers FF4 and FR3 detected the wild-type allele and generated a 380-bp fragment; primer MR3 and FR3 amplify a 180-bp fragment of the trapped allele.
For genotyping embryos at 1-, 2- and 4-cell stage, pregnant females were sacrificed approximately 12 h after mating and 1-cell stage embryos were collected and cultured in M16 medium at 37°C and 5% CO2. In the case of the fertilized eggs, the cumulus cells were removed by hyaluronidase treatment (Hogan et al., 1994) prior to washing. Embryos at 1-, 2- and 4-cell stage were then transferred individually into drops of acidic Tyrodés solution for 2–3 min to remove the zona pelucida and then into drops of trypsin for 3–5 min to separate polar bodies from embryos. Embryos were washed in phosphate-buffered saline (PBS), placed in 10 µl TE (10 mM Tris-HCl, 1mM EDTA, pH 8.0) and then lysed by repeatedly freezing on dry ice and thawing at 90°C. The entire lysate was used for the PCR genotyping. Embryos at morula (embryonic Day 2.5) and blastocyst stage (E3.5) were collected and placed individually into 10 µl TE and then lysed. The entire lysate was used for the PCR genotyping.
Northern blots and RT–PCR
Total RNA was extracted from cell cultures and tissues using a Quiagen RNA kit (Quiagen, Hilden, Germany). For northern blot analysis, 15 µg of RNA were separated in 1.2% agarose gels containing 2.2 M formaldehyde, transferred to nylon membranes (Amersham Biosciences, UK) and hybridized with the 32P-labelled cDNA probe at the same conditions as used for Southern blot hybridizations.
Embryos (100/assay) were isolated as described above, frozen in an ethanol/dry ice bath, and stored at –80°C. After addition of 10 µg rRNA (Roche), 200 µl of denaturing solution was used to lyse cells and extract RNA using Quiagen RNA kit (Quiagen).
RT–PCR assays were performed using total RNA and the One Step RT–PCR kit (Quiagen). Primer sequences to amplify the Faf1 transcripts in early embryonic stages were FF2: 5'-ATGAGCTTCAGATACCTGTGC-3' and FR2: 5'-GCATGGCATCATCTACCCTGA-3', and those to amplify cDNA fragment of Faf1-trapped transcript were IRESF1: 5'-TAACAAAGAGGACAAGCGGCCT-3' and UBAR1: 5'-AGCTCAAATGTCTTCCTGTTGTTG-3'. The Gapdh transcripts were amplified with the primers 5'-CCTGCTGGATTACATCAAAGCACTG-3' and 5'-GTCAAGGGCATATCCTACAACAAAC-3'. RT–PCR products were analysed in 1.5% agarose gels. Amplified cDNA fragment of Faf1-trapped transcripts was cloned into the pGEMT easy vector and sequenced.
Immunoblotting and immunohistochemistry
Proteins were isolated from various mouse tissues using RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to manufacture
s instruction. An aliquot of 25 µg of protein was separated on a 12% SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. Blots were blocked with 5% skim milk in PBS before incubation with the primary antibodies in PBS with skim milk overnight at 4°C. After the washing step, bound antibodies were detected using horse-radish peroxidase-conjugated rabbit anti-goat and anti-mouse immunoglobulin G (Sigma, St Louis, MO, USA) and enhanced chemiluminescence (Pierce Chemical, Bonn, Germany). The primary antibodies and dilutions used were goat anti-FAF1 (M-20, Santa Cruz Biotechnology) at 1:1000 and monoclonal anti- 
tubulin (Sigma).
For immunohistochemistry, tissues were fixed in Bouin‘s solution or in 4% paraformaldehyde/PBS, dehydrated, embedded in paraffin and sectioned at 7 µm. Sections were dewaxed, rehydrated, preincubated for 1 h with 5% horse serum in 0.05 Triton X-100/PBS, incubated overnight at 4°C with 1:250 diluted FAF1 antibody, washed three times in 0.05% Triton X-100/PBS, and then incubated with alkaline phosphatase-conjugated rabbit anti-goat antibody (1:1000) for 30 min at room temperature. After washing in PBS for 15 min, the immunoreactivity was detected by incubating the sections with a solution containing fast red TR/naphthol AS-MX phosphate tablets (Sigma).
Preimplantation embryos were fixed in buffered 4% paraformaldehyde for 15 min, followed by 0.25% Triton X-100/PBS treatment for 10 min. After pretreatment with 1% heat inactivated horse serum in PBS (antibody incubation buffer) for 60 min, embryos were incubated overnight at 4°C with the anti-FAF1 antibody, and washed three times in 0.25% Triton-X100/PBS for 15 min. Embryos were incubated for 60 min with the fluorescent-tagged rabbit anti-goat antibody diluted (1:1000). After three further washes with PBS, slides were mounted in 4', 6-diamidino-2-phenylindole mounting solution (Vector Laboratories, Burlingame, CA, USA). Slides were examined using a BX60 microscope (Olympus, Hamburg) with fluorescence equipment and an analysis software program (Soft imaging system, Münster, Germany).
To determine specificity of the primary antibody, M-20 antibody was pretreated with the blocking peptide (Santa Cruz Biotechnology) for 1 h at room temperature prior to incubation with western blots or oocytes/embryos.
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Expression of FAF1 in germ cell and early embryonic stages
To study the expression pattern of Faf1, northern and western blot analyses were performed using RNA and protein from different tissues of adult mouse. The Faf1 cDNA probe containing a coding sequence detected only 2.8-kb transcripts in all tissues studied (Fig. 1A). In western blots, an anti-FAF1 antibody against the C-terminus of the protein detected a 74-kDa FAF1 protein in all tissues and a further 49-kDa protein in testicular extract. The level of the 49-kDa isoform in testis is higher than the 74-kDa protein (Fig. 1B). The specificity of the antibody that recognizes a 74- and a 49-kDa protein was assessed by a competition assay. Excess antigen incubated with the antibody eliminated the 74- and 49-kDa bands (Fig. 1C).
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To evaluate the expression of Faf1 during post-natal development of testis and to determine the testicular cell types expressing the 49-kDa protein, we performed RNA and protein analyses. The first wave of male germ cell development initiated after birth yields fully developed spermatozoa in 35-day-old mice (Bellve et al., 1977). At post-natal Day 17, the mouse testes contain spermatogonia and spermatocytes, and at Day 21 the first wave of spermatids is observed. As shown in Fig. 1D, the Faf1 transcripts can be detected during the first three weeks of post-natal development. At post-natal Day 30, the level of Faf1 transcripts increases and remains high thereafter (Fig. 1D). In terms of protein, the 74-kDa isoform was expressed uniformly throughout testicular development while the expression of the 49-kDa isoform was detected at Day 30. Thereafter, we observed an increasing amount of the testis-specific isoform (Fig. 1E). The simultaneous increase of Faf1 transcript and the first appearance of the 49-kDa protein suggest that the 49-kDa isoform is a result of post-translational modification of the 74-kDa FAF1 precursor in haploid spermatids. We also examined the presence of 49-kDa FAF1 in the testes of mouse mutants, in which spermatogenesis is arrested at different stages. As expected, the 49-kDa protein was found in testes of qk/qk mutant mice (in which spermatogenesis is arrested at the spermatid stage) (Fig. 1F), whereas 49-kDa protein could not be detected in the testes of W/Wv mutant mice (which lack all germ cells) or in the cryptorchid testes of Insl3–/– mutant mice (in which spermatogenesis is arrested at the stage of pachytene spermatocytes). To investigate FAF1 expression during germ cells differentiation, immunohistochemistry was undertaken in testis sections and in germ cell suspensions of 9-week-old animals. In Sertoli and Leydig cells of wild-type males, FAF1 immuno-positive staining was barely detectable. Immuno-positive reactions were observed in all germ cells, but the most intense immunoreaction was found in elongated spermatids (Fig. 1G). In preparations of germ cell suspension, a high level of FAF1 immunostaining was found in cytoplasm of elongated spermatids and in giant cells containing multiple nuclei. No FAF1 expression was discernable in the mature spermatid (Fig. 1I). High accumulation of FAF1 was also observed in multinuclear giant cells in cryptorchid testis of Insl3 deficient mice (Fig. 1H).
Expression of FAF1 protein in oocytes and early cleavage embryos
In the adult ovary, FAF1 was detected in the cytoplasm of oocytes in all follicle stages (Fig. 2A and B). Indirect immunofluorescent labelling with the anti-FAF1 antisera was used to assess protein expression in oocytes as well as in early preimplantation embryos. Consistent with our immunohistochemistry results (Fig. 2B), FAF1 protein was localized in the cytoplasm of unfertilized oocytes (Fig. 2C). FAF1 protein assessment after fertilization demonstrated that the protein persists in early zygotes (Fig. 2D), as well as in early cleavage embryos at the 2-, 4- and 8-cell stages (Fig. 2E and F). Thus, FAF1 could theoretically function at any stages of oogenesis and in early embryos.
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Finally, we determined the preimplantation expression profile of Faf1 by RT–PCR on total RNA prepared from embryos at different preimplantation stages. To control for relative abundance of Faf1, we included RT–PCR for ubiquitously expressed Gapdh gene. Faf1 transcript could not be detected in 1- and 2-cell embryos. From the 4-cell to blastocyst stage, Faf1 mRNA increased dramatically, consistent with the expression of the zygotic Faf1 (Fig. 2G).
Generation and characterization of the Faf1-trapped gene
In a large-scale gene trap-screening programme to identify mouse lines, in which the trapped gene is prominently expressed in the gonads, we have generated and characterized mouse line 2C98. Breeding of 15-trapped males and females from F2 generation each with wild-type mice and genotyping their offspring with primers locating in the βGeo cassette of gene trap vector revealed that all animals of F2 generation were either wild type or heterozygous. These results suggest that the homozygous mutants die during embryonic development.
Using 5' rapid amplification of cDNA ends with primers constructed from the known vector sequences, we failed to identify a fusion transcript in RNA from adult testis. Therefore, a genomic library was constructed from the DNA of the 2C98 mouse line and screened with specific probes for the LacZ reporter gene. Sequence analysis revealed that the gene trap vector had integrated in the 17-kb long intron 7 of Faf1 (Fig. 3A). We referred to this mutation as Faf1GT. Southern blot hybridization of BamHI- and SstI-digested DNA from wild-type and Faf1-trapped mice with a probe located 3' of insertion site revealed that the insertion of the gene trap vector caused a shift in one restricted fragment in both digestions of gene trapped DNA (Fig. 3C). To evaluate whether Faf1 transcripts were affected by the gene trap integration, northern blot analysis was performed with RNA from different tissues of adult wild-type and Faf1GT/+ mice. Using cDNA probes localized 5' and 3' of the gene trap integration site, two transcripts, a 2.8-kb wild-type and a 3.0-kb fusion mRNA were detected in the tissues of Faf1GT/+ mouse (Fig. 3D). Hybridization of the 5' and 3' cDNA probes with the 3.0-kb fusion transcripts suggests a partial integration of the gene trap vector into 3.0-kb transcripts. Western blot analysis confirmed that in Faf1GT/+ mice, FAF1 expression was reduced by 
50% (Fig. 3E). To determine the molecular origin of the fusion transcript, RT–PCR analyses were performed using primers located in the IRES sequence of the gene trap vector and of exon 9 of Faf1. The sequence of amplified fragments showed that fusion transcripts, generated by proper joining of exon 7 with the SA site of the gene trap cassette had caused activation of the cryptic splice donor site which is located 115-bp downstream, and which splices to the SA of exon 8 (Fig. 3B). The 115-bp insertion creates a frameshift with premature termination of translation within the eighth exon of Faf1. Therefore, the resulting mRNA fails to encode functional FAF1 protein.
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Faf1 mutation results in early embryonic lethality
Genotyping of post-natal offspring from heterozygous mating by Southern blot and PCR analyses did not identify any homozygote Faf1GT/GT animals, and the ratio of wild type to heterozygote was 1:2 (Table I). These data are consistent with a homozygous lethal phenotype.
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To assess the consequences of the Faf1 mutation for embryonic development, embryos were collected from heterozygous intercrosses at different days of gestation. An analysis of E8.5, E12.5 and E14.5 embryos revealed that none of the 89 embryos genotyped were Faf1GT/GT (Table I). It is noteworthy that we did not observe a higher incidence of empty or resorbed deciduas at E8.5 in heterozygous intercrosses as compared to control crosses (data not shown). We concluded from this observation that Faf1GT/GT embryos die during the preimplantation period. We have then collected and cultured E0.5 embryos. Zona pellucida-free embryos at 1-, 2-, 4- and 8-cells stage were treated with trypsin to remove the polar bodies and then genotyped by PCR (Fig. 3F). At 1- and 2-cell stages, Faf1GT/GT embryos were detected (Table I), whereas no homozygous embryos were identified at 4- and 8-cell stage. Genotyping of E2.5 and E3.5 from heterozygous mating did not identify homozygous embryos at morula and blastocyst stage. To determine the causes of embryonic lethality, 2-cell embryos recovered from pregnant females obtained from heterozygous breeding were cultured for 24 h and developmental progress was observed. Of 48 cultured embryos, 39 embryos developed to 4-cell and morula stage, 4 were necrotic and 5 were arrested at 2-cell stage. Genotyping of the arrested 2-cell embryos showed that three of them were Faf1GT/GT. These results suggest that development of Faf1GT/GT embryos is arrested at 2-cell stage.
In order to rule out germ cell effects, the frequencies of genotypes in pups resulting from Faf1GT/+ x Faf1+/+ crosses involving both sexes are analysed. As seen in Table I, there was no under-representation of Faf1GT/+ pups either when the father or the mother contributed the Faf1GT allele. These results support the hypothesis that germ cell effects do not play a role for the observed lethality. Therefore, these data suggest that FAF1 is a required during preimplantation development.
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Discussion |
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We report here the expression pattern of Faf1 and the initial characterization of a mutant mouse line that has been generated from ES cells harbouring gene trap vector integrated in the Faf1 locus. The Faf1 is ubiquitously expressed as a 2.8-kb transcript, which most likely generates a 74-kDa protein in all adult mouse tissues as well as a testes-specific isoform with a molecular mass of 49-kDa. Expression analysis revealed that the increase of Faf1 transcripts correlates with the development of the post-meiotic haploid spermatids. In terms of protein levels, a uniform expression of the 74-kDa isoform was detected throughout testicular development. In contrast, expression of the 49-kDa isoform could be detected at Day 30. Thereafter, an increasing level of 49-kDa was observed. The coincidence of these events with the increase of Faf1 transcripts and the appearance of the 49-kDa isoform indicate that the 49-kDa isoform is a result of post-translational modification of the 74-kDa FAF1 precursor in spermatids. Immunohistochemistry revealed the most intense FAF1 immunoreactions in cytoplasm of elongated spermatids, while it was diminished in mature spermatids. FAF1 is prominent in multinuclear giant cells, which undergo cell death. This suggests that FAF1 is involved in the ubiquitin-dependent protein degradation pathway during spermatids differentiation and in apoptotic germ cells.
The VCR/p97 in vertebrates and its orthologues in yeast (Cdc48p) are involved in diverse cellular processes including protein degradation, membrane trafficking and cell cycle regulation (Woodmann et al., 2003). For its various functions, p97 forms a complex with different adaptors and cofactors. The UBX- and UBA-domain of FAF1 has been found to interact with VCP/p97 and multiubiquitinated substrates, respectively (Song et al., 2005). It seems more likely that FAF1 is an additional factor for the recruitment of VCP/p97 to the ubiquitinated proteins and that it regulates protein degradation in the ubiquitin proteasome pathway (Hartmann-Petersen et al., 2004; Schuberth et al., 2004; Neuber et al., 2005; Song et al., 2005). The disruption of orthologue gene encoding the interaction partner of FAF1 in yeast (cdc48) and Drosophila (TER94) is lethal (Moir et al., 1982; Leon and Mckearin, 1999). Recently, it has been shown that targeted disruption of p97 in mice leads to embryonic death at the preimplantation stage (Müller et al., 2007). The early embryonic lethality resulting from ablation of FAF1 and p97 in mouse indicates that FAF1-p97 protein complex play essential roles for cell growth and survival.
The FAF1 is evolutionarily conserved, but the fact that its in vivo function in mice was unknown, prompted us to characterize a Faf1-trapped mouse line. We found that FAF1-deficient embryos die at about the 2-cell stage, indicating that FAF1 is essential for cell viability and/or cell division. The ability of Faf1GT/GT to sustain cell division until the 2-cell stage can be interpreted as an effect of maternal FAF1 that could replace zygotic FAF1. Immunostaining revealed the presence of FAF1 protein in unfertilized oocytes and in all preimplantation stages, while the embryonic expression of Faf1 becomes appreciable at 4-cell stage as assessed by RT–PCR. The lethality of Faf1GT/GTembryos near the 2-cell stage corresponds to the onset of transcription and the increase of translation activity in the major phase of zygotic activation. Zygotic genome activation marks the passage from maternal to embryonic control of development, and represents the first crucial hurdle in the life of the individual (Schultz, 1993; Aoki et al., 1997; Schultz, 2002). Protein degradation is likely to be of major importance in early embryogenesis because of the need for each developmental stage to eliminate proteins that are transiently expressed. Degradation of maternal proteins by the ubiquitin proteasome pathway is also crucial during oocytes-to-embryo transition. The complex phenotypes observed in Caenorhabditis elegans zygotes that lack various components of ubiquitination machinery suggest that protein degradation regulates many development events during early embryogenesis. Indeed, RNA interference-mediated knockdown of Ubxn-3, the C. elegans homologue of Faf1 gene, leads to early embryonic lethality (Kamath et al., 2003; Fernandez et al., 2005). The failure of Faf1GT/GT mouse embryos to develop beyond the 2-cell stage differs from the semi-lethal phenotype of Drosophila caspar mutants. The casparP1 flies contain an EP-element insertion in the C-terminal part of protein-coding sequence of Drosophila Faf1. Expression analyses revealed that caspar mutant allele transcribes a truncated mRNA lacking the 3‘part of the transcript (Kim et al., 2006). Therefore, the caspar mutation may produce a hypomorphic allele for Faf1 that permits survival into adulthood.
In summary, we have characterized the expression pattern of FAF1 during spermatogenesis, oogenesis and early embryonic development, and demonstrated that it is essential for cellular viability. The early embryonic death and the limited number of Faf1GT/GT embryos available for study prevented us from defining more precisely its role in early embryonic cells. Future work, including the use of conditional gene targeting, should allow us to decipher the molecular mechanisms underlying its biological action.
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Funding |
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German Research Foundation (graduation Collage 242).
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Acknowledgements |
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We appreciate the contributions of Stephan Wolf in the breeding of gene trap mice. We thank C. Schwabe (University of South Carolina) for critical reading of the manuscript.
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Footnotes |
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These authors contributed equally to the paper. 
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Submitted on December 12, 2007; resubmitted on February 14, 2008; accepted on February 21, 2008.
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