RED CELLS

Targeted disruption of the murine , Fancg/Xrcc9

Yi Yang, Yanan Kuang, Rocio Montes De Oca, Tobias Hays, Lisa Moreau, Naifang Lu, Brian Seed, and Alan D. D’Andrea

Fanconi anemia (FA) is a human autoso- previously described Fancc؊/؊ and downstream Fancd2 in response -mal recessive cancer susceptibility disor- Fanca؊/؊ mice, the Fancg؊/؊ mice had to IR. Finally, Fancg؊/؊ mice had de der characterized by cellular sensitivity to normal viability and no gross develop- creased fertility and abnormal gonadal mitomycin C and ionizing radiation. Six mental abnormalities. Primary splenic histology. In conclusion, disruption of the FA (corresponding to subtypes A, lymphocytes, bone marrow progenitor Fancg gene confirms the role of Fancg in C, D2, E, F, and G) have been cloned, and cells, and murine embryo fibroblasts from the FA pathway. The Fancg؊/؊ mouse may the encoded FA interact in a the Fancg؊/؊ mice demonstrated sponta- be useful as an animal model for future common cellular pathway. To further un- neous breakage and in- gene therapy and cancer susceptibility Downloaded from http://ashpublications.org/blood/article-pdf/98/12/3435/1678703/h8230103435.pdf by guest on 28 September 2021 derstand the in vivo role of one of these creased sensitivity to mitomycin C and, studies. (Blood. 2001;98:3435-3440) human genes (FANCG), we generated a to a lesser extent, ionizing radiation. targeted disruption of murine Fancg and Fancg؊/؊ lymphocytes had a defect in the bred mice homozygous for the targeted FA pathway, based on their failure to allele. Similar to the phenotype of the activate the monoubiquitination of the © 2001 by The American Society of Hematology Introduction

Fanconi anemia (FA) is an autosomal recessive cancer susceptibil- observed. In addition, an increase in chromosome breaks in splenic ity syndrome characterized by multiple congenital anomalies and lymphocytes in response to bifunctional alkylating agents was observed. progressive bone marrow failure.1,2 FA patients develop several In both models, FanccϪ/Ϫ mice had germ cell defects and decreased types of cancers, including acute myeloid leukemias and squamous fertility. Unlike human FA patients, the FanccϪ/Ϫ mice had no obvious cell carcinomas of the head and neck.3 FA cells are sensitive to gross developmental abnormalities or cancer susceptibility. To date, the DNA cross-linking agents, such as mitomycin C (MMC) and, to a Fancc murine knockout model has been useful in examining (1) the role lesser extent, ionizing radiation (IR).4,5 Based on somatic cell of the Fancc gene in the physiologic response of hematopoietic cells to fusion studies, FA is composed of 8 distinct complementation DNA damage,24,25 (2) the in vivo effects of inhibitory cytokines on FA groups.6,7 Six of the human FA genes, including the genes for marrow cells,23,26,27 and (3) the efficacy of gene therapy.28-30 FANCA,8,9 FANCC,10 FANCD2,11 FANCE,12 FANCF,13 and In principle, targeted disruption of other murine FA genes, such FANCG,14 have been cloned. as the Fancg/xrcc9 gene, may provide additional insight to in vivo Recent studies have demonstrated that the 6 cloned FA proteins function. First, additional FA gene knockout models will allow a interact in a common cellular pathway.15 The FANCA, FANCC, side-by-side comparison of disease severity in different FA sub- FANCE, FANCF, and FANCG proteins assemble in a multisubunit types. Recent studies have suggested that the severity of FA, in nuclear complex.16-21 The FAprotein complex regulates the monoubi- terms of developmental abnormalities and hematologic defects, is quitination of the downstream FANCD2 protein, suggesting that dependent on FA subtype.31 For instance, FA-C and FA-G patients the complex is a multisubunit monoubiquitin ligase or regulates a exhibit more severe disease, while FA-A patients exhibit more mild ligase activity. When normal (non-FA) cells are exposed to disease, with later onset of bone marrow failure and hematologic DNA-damaging agents, such as IR, MMC, or UV light, FANCD2 malignancy. Whether mice with targeted disruptions of different is monoubiquitinated and targeted to nuclear foci containing the FA genes vary in disease severity remains untested. Second, BRCA1 protein.15 Disruption of this pathway leads to the character- conflicting studies suggest that some FA patients and cell lines are istic cellular and clinical abnormalities observed in FA. sensitive not only to mitomycin C (MMC) and diepoxy butane (DEB) In an attempt to understand the in vivo function of FAgenes, targeted but also to IR. Accordingly, murine knockout models may allow the disruptions of FA genes have been generated. Two murine models, systematic evaluation of differential drug and IR sensitivity. Third, the containing disruptions of the murine homolog of FANCC, have been availability of other FA gene disruptions will allow the generation and developed. Chen et al22 generated a disruption of exon 8 of Fancc, while characterizaton of mice with multiple FA gene knockouts. For instance, Whitney et al23 used to create a disruption of if 2 FAgenes function exclusively in the same cellular pathway, a double exon 9. In both models, spontaneous chromosome breakage was knockout should have the same phenotype as the single FA gene

From the Department of Molecular Biology, Massachusetts General Hospital; by RO1-AI27849 and UO1-HL66678. Department of Genetics, Harvard Medical School; Department of Pediatric Reprints: Alan D. D’Andrea, Dana-Farber Cancer Institute, Dept of Pediatric Oncology, Dana-Farber Cancer Institute; and Department of Pediatrics, Oncology, Harvard Medical School, 44 Binney St, Boston, MA 02115; e-mail: Children’s Hospital, Harvard Medical School; all of Boston, MA. [email protected]. Submitted March 15, 2001; accepted July 23, 2001. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby Supported by National Institutes of Health grants RO1-HL52725-04, marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734. RO1-DK43889-09, and PO1-HL54785-04 (A.D.D.). Y.Y. is supported by a postdoctoral fellowship from the Cancer Research Institute. B.S. is supported © 2001 by The American Society of Hematology

BLOOD, 1 DECEMBER 2001 ⅐ VOLUME 98, NUMBER 12 3435 3436 YANG et al BLOOD, 1 DECEMBER 2001 ⅐ VOLUME 98, NUMBER 12 knockout. In the current study, we have used homologous recombi- Alternatively, cells were cultured for 60 hours, exposed to IR (2 or 4 Gy, as nation to disrupt the murine Fancg gene and have analyzed the indicated), and allowed to recover for 12 hours before chromosome primary cells and tissues derived from FancgϪ/Ϫ mice. breakage or trypan blue exclusion (viability) analysis.

Murine hematopoietic progenitor assay Materials and methods Mononuclear cells were isolated from the femurs and tibiae of 4- to 6-week-old Fancgϩ/Ϫ or FancgϪ/Ϫ mice, as previously described. A total of Generation of Fancg-deficient mice and genotyping 2 ϫ 104 cells were cultured in 1 mL of MethoCult M3434 media (StemCell Technologies, Vancouver, BC, Canada) with or without MMC treatment. The murine Fancg gene was disrupted by replacing exons 2 to 9 with an Colonies were scored at day 7, when most of the colonies belong to the FRT-flanked neomycin cassette via homologous recombination in 129/ granulocyte-macrophage colony-forming unit or erythroid burst-forming SvJae embryonic stem cells. Mice homozygous for the Fancg mutation unit lineages. Each number was averaged from duplicate plates, and the within a mixed genetic background of 129/Sv and C57BL were generated data were derived from 2 independent experiments. following standard protocols. Mouse tail genomic DNA was prepared as 32 previously described and used as a template for polymerase chain reaction Immunoblotting of the murine Fancd2 protein Downloaded from http://ashpublications.org/blood/article-pdf/98/12/3435/1678703/h8230103435.pdf by guest on 28 September 2021 (PCR) genotyping. PCR reactions were assembled according to the manufacturer’s protocols (Roche, Indianapolis, IN) with primers Gex5F, Gex6R, Primary splenic lymphocytes, prepared as above, were cultured in RPMI Psv, and G40 (Figure 1) in the same reaction tube, at a final concentration of 1␮M medium plus 10% heat-inactivated fetal bovine serum for 72 hours. Cells each. The cycling conditions were 1 cycle of 94°C for 3 minutes; then 30 cycles were untreated or irradiated with IR (2, 4, 10, and 20 Gy). Cells were lysed, of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 45 seconds; followed and cellular proteins were electrophoresed, transferred to nitrocellulose, by 1 cycle of 72°C for 5 minutes. Mouse testis RNA was extracted using the SV and immunoblotted with a polyclonal antibody raised against human Total RNA Isolation System (Promega, Madison, WI). Fancg messenger RNA FANCD2.15 This antiserum cross-reacts with the Fancd2 protein in (mRNA) and ␤2-microglobulin mRNA were simultaneously amplified from 1 murine cells. ␮g total RNA by reverse transcriptase (RT)-PCR using the OneStep RT-PCR kit (Qiagen, Valencia, CA). The amplification conditions were 50°C for 30 minutes Flow cytometry for 1 cycle; 95°C for 15 minutes for 1 cycle; 94°C for 45 seconds, 60°C for 45 Lymphocytes isolated from thymus, spleen, and peripheral lymph nodes seconds, and 72°C for 1 minute for 30 cycles; and 72°C for 10 minutes for 1 were stained for T- or B-lymphocyte surface molecules with fluorescein cycle. Oligonucleotides (Figure 1A) used for PCR and RT-PCR were as follows: isothiocyanate–conjugated anti-CD3, anti-CD4, and anti-CD19 and phyco- Gex5F: 5ЈCCTCTGAGGATCTGCTACTACTGC3Ј. erythrin-conjugated anti-CD8, anti-CD44, anti-CD45RB, immunoglubulin Gex6R: 5ЈGTGTACACCTGGACTAACACGGAC3Ј. M, and anti-B220 (BD PharMingen, San Diego, CA). Stained cells were G40: 5ЈTGGCTAAATTCACTAAGTG3Ј. analyzed on a Coulter Epics XL flow cytometry system. Psv: 5ЈAAGGTTGGGCTTCGGAATCG3Ј. M2F: 5ЈGTATGCTATCCAGAAAACCCCTC3Ј. Histology M2R: 5ЈCATGTCTCGATCCCAGTAGACGG3Ј. Mice ovaries and testes were isolated and fixed in 4% paraformaldehyde Chromosome breakage assay for murine splenocytes and further processed by the core facility of the Department of Pathology at Massachusetts General Hospital. The specimens were photographed on a Splenocytes were prepared from 6-week-old mice of known Fancg Zeiss Axiplot. genotype. Briefly, the spleen was dissected, crushed in RPMI medium into a single-cell suspension, and filtered through a 70 ␮m filter. Red cells were lysed in hypotonic ammonium chloride. The remaining splenic lympho- cytes were washed in phosphate-buffered saline and resuspended in Results RPMI/10% fetal bovine serum plus phytohemagglutinin. Cells were tested Gene targeting and gross phenotype of Fancg؊/؊ mice for viability by the trypan blue exclusion assay. Cells were cultured for 24 hours in media and exposed to MMC or DEB for an additional 48 hours. We have disrupted the murine Fancg gene by replacing exons 2 to 9 with an FRT-flanked neomycin resistance cassette through homolo- gous recombination in murine embryonic stem cells (Figure 1A). Two independent embryonic stem cell clones with the desired mutation were injected into C57BL/6 blastocysts, and one germ-line transmission competent male chimera was obtained and bred with C57Bl/6 females to generate heterozygous (Fancgϩ/Ϫ) mice. Approximately 25% of the offspring of heterozygous breeders were homozygous (FancgϪ/Ϫ) as determined by genomic PCR (Figure 1B), indicating that there was no embryonic lethality associated with biallelic Fancg mutations. Moreover, no wild-type Fancg mRNA tran- scripts were detected in the testis of the FancgϪ/Ϫ mice by RT-PCR using primers amplifying exons 5 and 6 (Figure 1C). This result demonstrates that these mice are null mutants for the Fancg gene. Figure 1. Targeted disruption of the murine Fancg gene. (A) Schematic represen- tation of the murine Fancg gene showing 14 exons. The targeted allele has exons 2 to Mutant animals weighed the same as littermate controls, and no 9 replaced by the FRT-flanked neomycin cassette. There are translational stop macroscopic developmental abnormalities of the limbs or other codons in 3 reading frames 5Ј to the neomycin cassette to terminate any potential organ systems were detected (data not shown). upstream translation. Primers used for genotype and RT-PCR are indicated. (B) PCR genotype of mouse tail DNA using primers indicated in panel A. Primers Gex5F and Increased chromosome breakage and decreased survival of Gex6R amplify a 285 (bp) product specifically from the wild-type allele Fancg؊/؊ cells in MMC (WT), whereas primers Psv and G40 amplify a 580 bp product specifically from the mutant allele (MT). (C) RT-PCR of the wild-type Fancg mRNA using primers Gex5F and Gex6R from Fancgϩ/Ϫ testes but not from FancgϪ/Ϫ testes. A 360 bp RT-PCR Cytogenetic analysis demonstrated that primary cells (splenic lympho- Ϫ/Ϫ product of ␤2-microglobulin was used as an internal control (␤2M). cytes) from the Fancg mice had significantly higher DEB-induced BLOOD, 1 DECEMBER 2001 ⅐ VOLUME 98, NUMBER 12 TARGETED DISRUPTION OF THE Fancg/Xrcc9 GENE 3437

Figure 2. Primary splenic lymphocytes from Fancg؊/؊ mice have increased DEB- and MMC-induced chromo- some breakage. (A) Comparison of the number of aberrations per cell after treatment with the indicated amount of DEB or MMC. (B) Metaphase spreads of splenocytes from Fancgϩ/Ϫ and FancgϪ/Ϫ mice after 72 hours of in vitro growth. Cells were exposed to either no drug (ϩ0), MMC (20 ng/mL), or IR (4 Gy). Arrows indicate radial forms or breaks.

and MMC-induced chromosomal aberrations, particularly radial forms, splenocytes, FancgϪ/Ϫ splenocytes had a slight but reproducible compared with control mice (Figure 2A). Spontaneous aberrations were increase in IR sensitivity based on their IR dose-dependent Downloaded from http://ashpublications.org/blood/article-pdf/98/12/3435/1678703/h8230103435.pdf by guest on 28 September 2021 also increased in FancgϪ/Ϫ splenocytes. In addition, we established chromosome breakage (Figure 4A). primary skin fibroblast cultures (MEF cultures) from day 18 mutant and To further demonstrate the differential IR sensitivity of Fancgϩ/Ϫ control embryos (data not shown). Treatment of the mutant fibroblasts and FancgϪ/Ϫ splenocytes, we exposed cells to variable amounts of with MMC and DEB revealed chromosome breakage and other IR and measured cell viability by the trypan blue exclusion assay cytogenetic abnormalities, analogous to primary skin fibroblasts from (Figure 4B). By this assay, FancgϪ/Ϫ cells had decreased survival human FA patients (Figure 2B). following IR, confirming their enhanced IR sensitivity. The bone marrow of the FancgϪ/Ϫ mutant mice was grossly Disruption of the FA pathway in Fancg؊/؊ cells normal compared with the bone marrow of normal littermate controls (data not shown). Also, the peripheral blood count and Recent studies have demonstrated that the FA proteins cooperate in blood cell morphology of the FancgϪ/Ϫ mutant mice were grossly a common biochemical pathway leading to the generation of normal. There were no significant differences for the hematocrits of FANCD2/BRCA1 foci.15 Five of the cloned human FA proteins Fancgϩ/ϩ (47.6% Ϯ 0.2%), Fancgϩ/Ϫ (48.5% Ϯ 3.7%), and (FANCA, FANCC, FANCE, FANCF, FANCG) interact in a FancgϪ/Ϫ (52.3% Ϯ 0.2%) mice. However, when bone marrow multisubunit protein complex,17,20,21 and this complex is required progenitor cells were grown in methylcellulose culture in the for the DNA damage–inducible monoubiquitination of the down- presence of MMC, the FancgϪ/Ϫ progenitor cells had a decreased stream FANCD2 protein.15 We therefore tested the FancgϪ/Ϫ survival rate compared with normal littermate (ϩ/Ϫ) control splenocytes for the integrity of the FA pathway (Figure 5). marrow (Figure 3). For a MMC concentration of 30 ng/mL, no As previously described,15 the FA protein complex (Fanconi A/C/E/ FancgϪ/Ϫ progenitor colonies were observed. F/G protein complex) is required for the monoubiquitination of the ϩ/Ϫ Cellular sensitivity of Fancg؊/؊ cells to ionizing radiation FANCD2 protein (Figure 5A). Murine Fancg splenocytes express both forms of the Fancd2 protein (Fancd2-S and Fancd2-L) (Figure 5B, Recent studies suggest that FA patients and FA cells are also lanes 7-11). In contrast, FancgϪ/Ϫ splenocytes failed to express the sensitive to IR,5,30,33 although the relative IR sensitivity of FA cells Fancd2-L isoform either in the absence or the presence of IR (lanes from different FA subtypes is unknown. The human FANCG/ 12-16). The FancgϪ/Ϫ splenocytes did express the Fancd2-S isoform XRCC9 complementary DNA was originally cloned by its ability (lanes 12-16). Taken together, these results demonstrate that the FancgϪ/Ϫ to functionally complement the MMC- and IR-sensitive Chinese splenocytes have a disruption of the FA pathway. hamster cell line, UV-40, suggesting that the FANCG gene product Fancg؊/؊ mice have germ cell defects and decreased fertility may function directly in the normal cellular response to MMC or IR damage.34 We therefore tested the sensitivity of the FancgϪ/Ϫ Several attempts to inbreed the homozygous FancgϪ/Ϫ mice have primary splenocytes to IR (Figure 4). Compared with Fancgϩ/Ϫ not yielded any offspring over a period of 6 months. Cross- breeding of Fancgϩ/Ϫ and FancgϪ/Ϫ mice has resulted in a decreased frequency of pregnancies and reduced litter sizes, as compared with Fancgϩ/Ϫ inbreeds, suggesting that both male and

Figure 4. Differential IR sensitivity of Fancg؉/؊ and Fancg؊/؊ primary splenic lymphocytes. (A) Analysis of chromosome breakage following exposure to variable amounts of IR. Results shown are representative of 3 separate experiments. (B) Figure 3. Bone marrow progenitor cells from Fancg؊/؊ mice have increased Analysis of cell viability by the trypan blue assay following IR exposure. Based on this sensitivity to MMC. Mononuclear cells were isolated from bone marrow from assay, the Fancgϩ/Ϫ and FancgϪ/Ϫ cells had approximately equal viability (95%-98% Fancgϩ/Ϫ mice or FancgϪ/Ϫ mice. Cells were plated in methylcellulose in the viable) in the absence of IR exposure. Data shown are representative of 3 separate presence of the indicated concentration of MMC. Colonies were enumerated at day 7. experiments. 3438 YANG et al BLOOD, 1 DECEMBER 2001 ⅐ VOLUME 98, NUMBER 12

In addition, we tested protein extracts from the testes of FancgϪ/Ϫ mice and littermate controls for expression of the Fancd2 protein (Figure 5B, lanes 17, 18). Interestingly, the FancgϪ/Ϫ mutant testis expressed only the short (nonubiquitinated) isoform of the Fancd2 protein (lane 18), while the Fancgϩ/Ϫ sibling control testis expressed both Fancd2-S and Fancd2-L isoforms (lane 17). Taken together, these data show that an intact FA pathway and the activation of Fancd2 to the monoubiquitinated isoform of Fancd2 correlates with the ability of the germ cells to differentiate into mature spermatocytes.

Discussion Downloaded from http://ashpublications.org/blood/article-pdf/98/12/3435/1678703/h8230103435.pdf by guest on 28 September 2021 In the current study, we used homologous recombination to disrupt the murine Fancg gene and to generate a murine model of FA subtype G. The cellular and gross phenotype of the FancgϪ/Ϫ mice was similar to the phenotype observed for the previously described FanccϪ/Ϫ mice.22,23 Primary cells from the FancgϪ/Ϫ mice demon- strated spontaneous chromosome breakage that was increased in response to the DNA cross-linking agents, MMC and DEB. While the FancgϪ/Ϫ mice had few gross phenotypic abnormalities, the mice were infertile and had clear histologic abnormalities of the gonads. While the bone marrow cellularity and peripheral blood ery- throid mass (hematocrit) for the FancgϪ/Ϫ mice were normal, the bone marrow progenitor cells were sensitive to MMC. Decreased survival of FancgϪ/Ϫ bone marrow progenitor cells was observed in methylcellulose culture. Similar MMC sensitivity has previously been reported for bone marrow from FanccϪ/Ϫ mice. Figure 5. Primary splenic lymphocytes from FA-G mice are defective in Taken together, our data suggest that the FancgϪ/Ϫ mice exhibit IR-inducible Fancd2 monoubiquitination. (A) Schematic representation of the minor hematologic abnormalities characteristic of FApatients during the human FA pathway. The FA protein complex, containing the FANCA, FANCC, Ϫ/Ϫ FANCF, FANCG, and perhaps FANCE proteins, is required for the monoubiquitina- preanemic phase. However, in contrast to human FA patients, Fancg tion of the FANCD2-S protein (155 kd) to the FANCD2-L protein (162 kd). mice do not develop progressive bone marrow failure, at least not during Monoubiquitination is increased in response to DNA damage by IR. Ubiquitinated the first year of life. The biological significance of the difference (Ub) FANCD2 is targeted to foci containing the BRCA1 protein and other proteins involved in DNA repair. (B) Primary splenic lymphocytes (splenocytes) were isolated between human and murine FA is unclear. It is possible that additional from mice with the indicated Fancg genotype. Cells were untreated or exposed to IR redundant antiapoptotic signaling pathways exist in the murine system, (2, 4, 10, or 20 Gy), as indicated. Cell lysates were prepared, and total cellular which partially compensate for the loss of the FA pathway in murine proteins were electrophoresed, transferred to nitrocellulose, and immunoblotted with bone marrow cells. an anti-Fancd2 antiserum. This polyclonal antiserum cross-reacts with the human and murine Fancd2 protein and also recognizes a nonspecific 150 kd protein in both It will be interesting to determine whether in vivo administra- human and murine lysates, as indicated. Alternatively, whole tissue extracts were tion of low doses of MMC to the FancgϪ/Ϫ mice versus sibling prepared from testes (lanes 17,18) from a Fancgϩ/Ϫ mouse or a FancgϪ/Ϫ mouse. control mice will result in bone marrow failure. Also of interest will Cells examined were PD7 (wild-type human lymphoblasts), PD20 (human FA-D2 lymphoblasts), Ba/F3 (murine interleukin-3–dependent lymphocytes), or murine be whether retroviral gene transfer of the wild-type human FANCG splenocytes (lanes 7-16). Both FANCD2-S and FANCD2-L isoforms are absent from complementary DNA corrects the MMC sensitivity of the bone the PD-20 (FA-D2) lymphoblasts, as previously described. marrow progenitors, leading to enhanced survival of transduced cells in vivo. female FancgϪ/Ϫ homozygote mutant mice have impaired fertility The primary splenic lymphocytes from the FancgϪ/Ϫ mice also had (data not shown). increased sensitivity to IR, based on the chromosome breakage assay To investigate the cause of the infertility, we performed and survival studies. Increasing evidence suggests that FA cells are measurements and histologic evaluation of the reproductive organs mildly sensitive to IR, although they are more sensitive to DNA of 6- to 8-week-old mice (Figure 6). The ovaries of FancgϪ/Ϫ cross-linking agents. For example, nonmyeloablative doses of IR have mutant mice were abnormal compared with those of littermate recently been shown to enhance the selection of wild-type hematopoi- controls. Mutant ovaries were almost completely devoid of follicles etic cells over FA hematopoietic cells in murine competitive repopula- (Figure 6A). The testes of FancgϪ/Ϫ mutant mice were also tion models.30 The IR sensitivity of primary FancgϪ/Ϫ cells in our study abnormal. The testicular weight of FancgϪ/Ϫ homozygote males 6 is consistent with other recent studies describing the FA molecular weeks and older was markedly reduced (218 Ϯ 47 mg per testis) pathway.15 IR activates the dose-dependent and time course–dependent compared with littermate Fancgϩ/Ϫ heterozygote controls (575 Ϯ 81 monoubiquitination of the FANCD2 protein in normal (non-FA) cells. mg per testis, P Ͻ .01). Similar to the testicular histology observed Monoubiquitinated FANCD2 is targeted to BRCA1 nuclear foci, which for the FanccϪ/Ϫ animal, the FancgϪ/Ϫ testis had a mosaic pattern appear to play a role in the repair of DNA double-strand breaks. of the seminiferous tubules, with the appearance of both normal Interestingly, the FA protein complex, including the FANCA, FANCC, and abnormal tubules. The abnormal tubules were devoid of FANCE, FANCF, and FANCG proteins, is required for the IR- spermatocytes and contained only Sertoli cells (Figure 6B). dependent monoubiquiitination of FANCD2. Human FA cells, defective BLOOD, 1 DECEMBER 2001 ⅐ VOLUME 98, NUMBER 12 TARGETED DISRUPTION OF THE Fancg/Xrcc9 GENE 3439

Figure 6. Fancg ؊/؊ mice have germ cell defects and decreased fertility. (A) Histology of mutant and control ovaries (hematoxylin and eosin staining). Ovary of an 8-week- old mutant or control mouse. The mutant ovary was almost completely devoid of follicles (iii, ϫ 60; iv, ϫ 125). The control ovary had an abundance of developing follicles at various stages (i, ϫ 60; ii, ϫ 125). (B) Histology of mutant and control testis (hematoxylin and eosin staining). A mosaic pattern of seminiferous tubules devoid of germ cells and normal tubules can be seen in the mutant testis (iv, ϫ 250; v and vi, ϫ 320). A control testis is shown at the same magnification (i, ii, iii). Arrows indicate Sertoli cells. Downloaded from http://ashpublications.org/blood/article-pdf/98/12/3435/1678703/h8230103435.pdf by guest on 28 September 2021

in the genes encoding any one of these FA proteins, fail to form Fancd2 protein. Recent studies demonstrate that the activated FANCD2/BRCA1 foci in response to IR, perhaps accounting for the IR human FANCD2 protein normally binds to meiotic sensitivity of the FancgϪ/Ϫ cells observed in the current study. in the axial (unsynapsed) regions of synaptonemal complexes.15 Other mouse models with enhanced IR sensitivity have defects in the FANCD2 binding correlates with the increase in DNA double- immune system. Specifically, mice with targeted disruptions of the Ku, strand breaks, resulting from crossing-over events during . DNA-PK, or ATM gene35-37 have cellular and humoral immunodefi- Based on this model,15 a disruption of the FA pathway, caused by a ciency. We therefore analyzed T cells and B cells from FancgϪ/Ϫ mice targeted disruption of the Fancg gene, may prevent normal Fancd2 versus Fancgϩ/Ϫ sibling controls (data not shown). For single-cell activation and may block the normal binding of Fancd2 to meiotic suspensions prepared from thymus, spleen, or peripheral lymph nodes, chromosomes in the FancgϪ/Ϫ animals. Absence of Fancd2 there was no difference in the expression of T-cell surface antigens staining of meiotic chromosomes may lead to accelerated apoptosis (CD3, CD4, CD8) or B-cell surface antigens (CD19, immunoglobulin of differentiating spermatocytes. M, B220) between FancgϪ/Ϫ and Fancgϩ/Ϫ mice. Consistent with these Finally, the similarity between the FanccϪ/Ϫ mouse23,38 and the studies, human FA patients are not immunodeficient and appear to have FancgϪ/Ϫ mouse in this study provides further genetic evidence of normal immunoglobulin diversity. a common FA molecular pathway.15,39 Previous studies have The FancgϪ/Ϫ mice are infertile, and the gonads from these demonstrated that FANCC and FANCG interact in a nuclear animals have striking pathology. Ovaries from the FancgϪ/Ϫ protein complex, along with other FA proteins (FANCA and females are small and have decreased cellularity. Testes from the FANCF). FANCE is probably a subunit of this nuclear complex as FancgϪ/Ϫ males are small and have decreased cellularity and well, based on in vitro binding studies. Other proteins may also be spermatogenesis. While the molecular basis of this gonadal histol- part of this nuclear complex, such as the recently cloned FANCC- ogy is unknown, it may result from a defect in the activation of the interacting protein, FAZF.40 The FA protein complex (A/C/E/F/G) 3440 YANG et al BLOOD, 1 DECEMBER 2001 ⅐ VOLUME 98, NUMBER 12 is required for DNA damage–inducible monoubiquitination and require the generation of mice with double or triple gene disrup- activation of the downstream FANCD2 protein. Activated FANCD2 tions. Also, it will be interesting to determine whether targeted interacts with chromatin and may be required for DNA repair. That disruption of Fancd2, the putative substrate of the FA protein disruption of the Fancc or Fancg genes yields the same phenotype complex, yields a similar phenotype to the Fancc, Fanca, and suggests that the major (or sole) function of the Fancc and Fancg Fancg knockout models. proteins is to cooperate in the FA protein complex. For instance, if the Fancc or Fancg proteins had additional functions beyond their role in the FA protein complex, one might expect additional unique Acknowledgments phenotypic characteristics for the FanccϪ/Ϫ and FancgϪ/Ϫ knock- out animals. Disruption of the murine Fanca gene also results in a We thank En Li for providing 129/SvJae embryonic stem cells; similar phenotype,41 further supporting a unified function of these Ramnik Xavier and Robin Mayfield for their help in histology proteins (A/C/G) in the FA protein complex. Whether any of the FA analysis; and Stephen Conley and Michelle Forestall for preparing genes have additional functions outside of the FA pathway will the histology slides. Downloaded from http://ashpublications.org/blood/article-pdf/98/12/3435/1678703/h8230103435.pdf by guest on 28 September 2021 References

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