Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12412-12417, October 1996 Genetics Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality (insertional mutagenesis/retroviruses/protein degradation) KLAUS HARBERS*t, URSULA MULLER*, ANJA GRAMS*, EN LID§, RUDOLF JAENISCHt, AND THOMAS FRANZI *Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie an der Universitat Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany; tWhitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142; and lAnatomisches Institut der Universitat Hamburg, Universitatskrankenhaus Eppendorf, Martinistrasse 52, D-20251 Hamburg, Germany Communicated by Alexander Varshavsky, California Institute of Technology, Pasadena, CA, July 29, 1996 (received for review May 15, 1996) ABSTRACT Ubiquitin-conjugating enzymes (E2 or Ubc) for recognition of specific proteolytic substrates and at least in constitute a family of conserved proteins that play a key role in one case have been shown to be directly involved in the final ubiquitin-dependent degradation of proteins in eukaryotes. We transfer of ubiquitin to a substrate protein (8). E2 enzymes are describe here a transgenic mouse strain where retrovirus inte- encoded by a multigene family and several members of this gration into an Ubc gene, designated UbcM4, results in a reces- family have been isolated from various organisms including sive-lethal mutation. UbcM4 is the mouse homologue of the plants. The function of E2 enzymes has been studied most previously described human UbcH7 that is involved in the in vitro extensively in yeast by usiing a genetic approach. Mutations in ubiquitination of several proteins including the tumor suppres- individual genes for dlifferent yeast E2 enzymes revealed that sor protein p53. The provirus is located in the first intron of the these proteins participate in a wide variety of essential cellular gene. When both alleles are mutated the level of steady-state functions (1). In Drosophila an E2 enzyme has been shown to mRNA is reduced by about 70%o. About a third of homozygous be involved in nervous system development (9, 10). Based on mutant embryos die around day 11.5 of gestation. Embryos that these results it is most likely that in higher organisms, including survive that stage are growth retarded and die perinatally. The man, mutations in the genes coding for the different enzymes lethal phenotype is most likely caused by impairment ofplacenta of the ubiquitin-conljugating system will contribute to abnor- development as this is the only organ that consistently showed mal development and disease. Indirect evidence for such a pathological defects. The placental labyrinth is drastically re- relationship has accumulated over the past years (11). duced in size and vascularization is disturbed. The UbcM4 mouse No mouse mutants with spontaneous or experimentally mutant represents the first example in mammals of a mutation induced alterations have been previously described that help to in a gene involved in ubiquitin conjugation. Its recessive-lethal clarify the role of any of the E2 genes during differentiation phenotype demonstrates that the ubiquitin system plays an and development. In the present report we describe a trans- essential role during mouse development. genic mouse mutant where integration of a retrovirus into an E2 gene, designated UbcM4, results in a recessive-lethal phe- Protein degradation is an important process bywhich cells control notype. Homnozygous embryos die in utero most likely as a the activity of normal proteins and avoid the potentially toxic result of impaired placenta development. effects of aberrant proteins. A major pathway for selective breakdown of proteins in eukaryotes is the ubiquitin/proteasome MATERIALS AND METHODS system in which proteins are conjugated to the polypeptide Derivation of A6 Mouse Strain. Cells of the embryonal stem ubiquitin and, thus tagged, are degraded by the proteasome (ES) cell line JI (12) were intfected by growing on a monolayer of complex (1-3). Substrates of this pathway include cyclins, cyclin- x-ray irradiated Psi-2 cells producing niplO virus (13) in ES cell dependent kinase inhibitors, transcription factors, the tumor culture medium containing 8 ,rg/ml polybrene. After coculture for suppressor protein p53, and a variety of oncoproteins (2). Very 48 h, ES cells were trypsinized and selected in medium containing recent evidence suggests that ubiquitination of membrane-bound G418 (125 p,g/ml of active component) for 10 days. G418-resistant receptors might also serve as a signal for degradation via the colonies were picked and expanded. Cell lines were analyzed by lysosomal pathway (4). Although ubiquitin conjugation is mainly Southern blot analysis for the presence of proviral DNA. ES cell thought to tag proteins for destruction, several lines of evidence lines with single copy proviral genome were injected into BALB/c suggest that it might also be involved in other cellular functions. or C57BL/6 blastocysts, and injected embryos were transferred This is indicated, for example, by the presence of stable ubiquiti- into uteri of pseudopregnant F1(C57BL/6J x CBA) foster moth- nated proteins, such as histones (5), and by the existence of ers as described (12). Of ninle independent ES cell lines injected, enzymes that remove ubiquitin from conjugates and therefore six contributed to the germ line of the mouse. Two of the six make this modification reversible (6). Reversible ubiquitination transgenic lines, A6 and A8, resulted in recessive defects in the might have a role in modulating the function of target proteiIns homozygotes. The inbred A6 mouse straini was obtained by similar to protein phosphorylation. breedinig a male chimera with a female 129/Sv mouse. The 129/Sv Attachment of ubiquitin to cellular proteins is carried out by strain was also used for all subsequent breedings in our colony. The the sequential action of three classes of enzymes, ubiquitin- genotyping was initially done by quantitative Southern blotting of activating enzymes (referred to as El or Uba), ubiquitin- tail DNA. After-the provirus flanking probe (5' fA6 in Fig. 2) was conjugating enzymes (E2 or Ubc), and ubiquitin-protein li- obtained all genotypes were confir-med by qualitative Southern gases (E3 or Ubr) (7). In an initial activating step the El blots and/or by PCR analysis (see below). Embryos were obtained enzyme forms a thiolester bond with the C terminus of ubiquitin, which is then transferred to a specific cysteine Abbreviations: E2 or Ubc, ubiquitin-conjugating enzyme; ES, embry- residue of the E2 enzyme. Although E2 enzymes can directly onal stem; LTR, long terminal repeat; Mo-MuLV, Moloney murine donate ubiquitin to proteins, E3 enzymes are typically required leukemia virus; RACE, rapid amplification of cDNA enids. Data deposition: The sequence reported in this paper has been deposited in the GenBank data base (accession no. X97042). The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in §Present address: Cardiovascular Research Center, Massachusetts accordance with 18 U.S.C. §1734 solely to indicate this fact. General Hospital, Charlestown, MA 02129. 12412 Downloaded by guest on September 24, 2021 Genetics: Harbers et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12413 from timed matings; the day of plug detection was counted as day binant retrovirus mplO (13). This virus is a replication-defective 0.5 of gestation. derivative of the Mo-MuLV and carries a neomycin resistance Molecular Cloning. The proviral integration site. subsequently gene under the transcriptional control ofthe Mo-MuLV LTR. The referred to as A6 locus, was cloned using standard methods (14). A6 mouse strain carries one copy of the provirus in its germ line. Initially a celltular DNA fragment flanking the 5' end of the mplO Animals heterozygous for provirus integration at the A6 locus did provirus was cloned by inverse PUCR (15). This fragment, desig- not display any overt abnormalities during development and nated 5' fA6 (see Fig. 2), was subcloned in the Bluescript vector postnatal life. However, crosses between heterozygous parents (Stratagene). To obtain phage A clones representing the A6 locus failed to produce homozygous mutant offspring at weaning, in- from wild-type mice, a 129Sv mouse genomic library in AFIXII dicative of lethality at an earlier age (Table 1). Embryos were (Stratagene) was screened with a radiolabeled probe derived isolated at different stages of gestation and genotyped by PCR from fragment 5' fA6. Positive plaques were purified by further analysis of yolk sac DNA as described below. Homozygous A6 rounds of screening. For fine nmappiing and structural analyses, embryos were recovered in the expected proportions between days subfragments from the A clonies were subcloned according to 10.5 and 19.5 of gestation (counting the day of vaginal plug as day standard procedure. 'Tlhe physical map of the A6 locus shown in 0.5). However, the proportion of normally developed homozygous Fig. 2 was obtained from Southern blot analyses of wild-type and embryos decreased with age, accompanied by an increasing num- mutant genomic DNA as well as from restriction analyses of the ber of embryos that were dead, or alive but with clear signs of A and plasmid