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Inactivation of Murine Usp1 Results in Genomic Instability and a Fanconi Anemia Phenotype

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Inactivation of Murine Usp1 Results in Genomic Instability and a Fanconi Anemia Phenotype CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Developmental Cell Short Article Inactivation of Murine Usp1 Results in Genomic Instability and a Fanconi Anemia Phenotype Jung Min Kim,1 Kalindi Parmar,1 Min Huang,1 David M. Weinstock,2 Carrie Ann Ruit,1 Jeffrey L. Kutok,3 and Alan D. D’Andrea1,* 1Department of Radiation Oncology 2Department of Medical Oncology Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA 3Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2009.01.001 SUMMARY FANCJ). The downstream FA genes are also breast cancer susceptibility genes (Grompe and van de Vrugt, 2007). Fanconi anemia (FA) is a human genetic disease Recent studies have indicated that the FANCD2/FANCI characterized by chromosome instability, cancer complex is deubiquitinated by the ubiquitin-specific protease, predisposition, and cellular hypersensitivity to DNA USP1 (Cohn et al., 2007; Huang et al., 2006; Nijman et al., crosslinking agents. The FA pathway regulates the 2005a). USP1 is not an FA gene per se, since no human FA repair of DNA crosslinks. A critical step in this patients harboring mutations in the USP1 gene have been iden- pathway is the monoubiquitination and deubiquitina- tified. Disruption of the Usp1 gene in chicken cells (DT40) results in crosslinker hypersensitivity, and the chicken Usp1 and Fancl tion of FANCD2. Deubiquitination of FANCD2 is genes are epistatic for crosslink repair (Oestergaard et al., 2007). mediated by the ubiquitin protease, USP1. Here, we The human genome encodes over 90 deubiquitinating demonstrate that targeted deletion of mouse Usp1 enzymes (DUBs) (Nijman et al., 2005b). In rare cases, disruption results in elevated perinatal lethality, male infertility, of a ubiquitin E3 ligase results in a similar outcome to disruption crosslinker hypersensitivity, and an FA phenotype. of a corresponding DUB (Nijman et al., 2005b), suggesting that Usp1À/À mouse embryonic fibroblasts had height- coupled ubiquitination and deubiquitination may be essential ened levels of monoubiquitinated Fancd2 in chro- for the function of some pathways. matin. Usp1À/À cells exhibited impaired Fancd2 foci In the current study, we disrupted the murine Usp1 gene. Inter- À/À assembly and a defect in homologous recombination estingly, Usp1 mice had a strong resemblance to FA mice (small size, infertility, mitomycin C [MMC] hypersensitivity, and repair. Double knockout of Usp1 and Fancd2 re- À/À sulted in a more severe phenotype than either single chromosome instability). In addition, Usp1 mice exhibited a higher rate of perinatal lethality and absence of male germ knockout. Our results indicate that mouse Usp1 cells. Our results indicate that Usp1 is required for Fancd2 foci functions downstream in the FA pathway. Deubiqui- assembly and contributes to homologous recombination (HR) tination is a critical event required for Fancd2 nuclear repair, suggesting a regulatory role of Usp1 in the FA pathway. foci assembly, release from chromatin, and function in DNA repair. RESULTS Perinatal Lethality and Growth Retardation in Usp1-Null INTRODUCTION Mutation To address the physiological role of Usp1, we performed a tar- The Fanconi anemia (FA) pathway mediates DNA repair and geted gene deletion of Usp1 in the mouse (see Figure S1 available promotes normal cellular resistance to DNA crosslinking agents online). Usp1+/À mice were grossly normal and fertile. Murine (Wang, 2007). The FA pathway is regulated by 13 FA proteins Usp1 was detected as two distinct protein products of different (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, and N) (Grompe and size in wild-type mouse embryonic fibroblasts (MEFs), and van de Vrugt, 2007). Eight of the FA proteins are assembled in Usp1+/À MEFs displayed approximately 2-fold reduction in a nuclear ubiquitin E3 ligase complex (FANCA/B/C/E/F/G/L/M), Usp1 protein level (Figure S1C). From Usp1+/À intercrosses, known as the FA core complex, which monoubiquitinates Usp1À/À mice were obtained at a low frequency (6%, instead of FANCD2 and FANCI (Dorsman et al., 2007; Sims et al., 2007; the expected 25%; Figure 1A). When analyzed during gestation Smogorzewska et al., 2007). The monoubiquitinated FANCD2/ (E13.5–E18.5) or after cesarean delivery at E19.5–E20.5, Usp1À/À FANCI complex is targeted to chromatin (Montes De Oca embryos, although significantly smaller than their wild-type et al., 2004), where it interacts, either directly or indirectly, with littermates (Figure 1C and data not shown), were present at the additional downstream FA proteins (FANCD1, FANCN, and expected Mendelian ratio. Thus, deletion of the Usp1 gene is 314 Developmental Cell 16, 314–320, February 17, 2009 ª2009 Elsevier Inc. Developmental Cell FA Phenotype in Usp1-Deficient Mice not embryonic lethal; rather, it led to perinatal lethality, since within 1–2 days after birth, up to 80% of the Usp1À/À mice were dead. Usp1À/À pups found dead displayed several abnormalities with incomplete penetrance; these included cyanosis, bilateral hydronephrosis, and hemorrhagic edema. When closely moni- tored at the moment of delivery, most of the Usp1À/À pups (75%) became progressively cyanotic and died within 2 hr of birth. Thus, cyanosis may be one of the causes of perinatal lethality in newborn Usp1À/À mice. The surviving Usp1-deficient mice were consistently smaller than their wild-type littermates, at weaning and as adults, suggesting that growth retardation occurred prenatally and persisted into adulthood (Figures 1B and 1C). Gonadal Dysfunction in Usp1-Deficient Male Mice Usp1À/À male mice were sterile. The Usp1À/À testis was strikingly smaller than that of wild-type or Usp1+/À littermates (Figure 1D and data not shown). Histology of adult testes from Usp1À/À mice revealed that their seminiferous tubules were markedly atrophic and mostly devoid of spermatogenic cells (e.g., spermatogonia, spermatocytes, spermatids, and spermatozoa; Figure 1E). Consistently, the epidydymis of Usp1À/À mice was devoid of spermatozoa (Figure 1E). We examined seminiferous tubules for cellular proliferation by Ki-67 immunostaining (Figure 1F). At 1 day after birth, Ki-67-positive cells were detected in wild-type and Usp1À/À tubules with similar frequency, and the intensity of Ki-67 staining in Usp1À/À tubules was even stronger than that of the wild-type tubules. At 3 and 16 weeks, however, cellularity of Usp1À/À tubules was greatly decreased, and remain- ing cells were mostly negative for Ki-67 staining (Figure 1F). Im- munostaining for cleaved Caspase 3 revealed that Usp1À/À tubules had a slightly increased frequency of apoptosis 1 day after birth (Figure 1G). However, the majority of Usp1À/À cells did not express cleaved Caspase 3, suggesting that apoptosis is not the major cause of testicular atrophy in Usp1À/À mice. Usp1À/À female mice showed a reduced fertility as compared with wild-type females. Usp1À/À ovaries had less oocytes than wild-type ovaries (8.5 ± 0.6 and 2.9 ± 0.3 per section for wild- type and Usp1À/À 12-week-old ovaries, respectively; five females each; Figure 1H). MMC Hypersensitivity and Enhanced Fancd2 and PCNA Monoubiquitination in Usp1-Deficient Cells There were no significant differences in cell cycle distribution between wild-type and Usp1À/À primary MEFs (either asynchro- nous or after release from synchronization in G0 by serum star- vation) (Figures S2A and S2B). As predicted from studies with (F) Detection of Ki-67 by immunohistochemistry in seminiferous tubules from wild-type and Usp1À/À mice. Magnification, 403. Figure 1. Perinatal Lethality, Growth Retardation, and Impaired (G) Detection of apoptosis by cleaved Caspase-3 staining in seminiferous Germ Cell Development in Usp1-Deficient Mice tubules from 1-day-old testes. Magnification, 633. (A) Viability of Usp1-deficient mice at different stages of development. (H) Usp1À/À ovaries (lower panel) had reduced numbers of oocytes (arrow- (B) Growth curve of wild-type (gray triangles) and Usp1À/À (filled triangles) head) compared with wild-type ovaries (upper panel), yet retained the follicle mice. (arrow) from which the oocyte had been released during ovulation. For the (C) Gross appearances of newborns and 2-week-old wild-type and Usp1À/À quantitative analysis, oocytes were counted from five females of each geno- mice. type at 12 weeks, and the number of oocytes was determined for each section (D) Comparison of testis size from 16-week-old wild-type and Usp1À/À mice. (8 sections per each female; total 40 sections). All visible oocytes (including (E) Sections of wild-type and Usp1À/À seminiferous tubules and epididymis small oocytes and immediately visible oocytes) were counted. Magnification, stained with hematoxylin and eosin. Magnification, 103. 53. Developmental Cell 16, 314–320, February 17, 2009 ª2009 Elsevier Inc. 315 Developmental Cell FA Phenotype in Usp1-Deficient Mice Figure 2. Hypersensitivity to DNA Cross- linking Agents in Usp1-Deficient Cells (A) Increased Fancd2-Ub and PCNA-Ub in Usp1- deficient cells. Wild-type and Usp1À/À MEFs were left untreated, or were treated with MMC (500 ng/ml for 20 hr) or UV (30 J/m2, harvested at 3 hr after irradiation). Cell lysates were immuno- blotted with indicated antibodies. (B) Survival rates of wild-type and Usp1À/À MEFs to MMC and UV treatment are determined as the percentage of viable cells relative to that for respective untreated cells. (C) MMC-induced chromosomal aberrations in wild-type (gray bars) and Usp1À/À (filled bars) MEFs following the treatment with MMC for 48 hr. The numbers of chromosomal aberrations (left panel) and radial forms (right panel) per meta- phase spread were scored. (D) Clonogenic survival assay of BM cells from wild-type (open squares), Usp1+/À (gray squares), Usp1À/À (filled squares), and Fancd2À/À (filled triangles) mice.
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