The hSSB1 orthologue Obfc2b is essential for skeletogenesis but dispensable for the DNA damage response in vivo
2012; Springer Nature; Volume: 31; Issue: 20 Linguagem: Inglês
10.1038/emboj.2012.247
ISSN1460-2075
AutoresNiklas Feldhahn, Elisabetta Ferretti, Davide F. Robbiani, Elsa Callén, Stephanie Deroubaix, Licia Selleri, André Nussenzweig, Michel C. Nussenzweig,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoArticle31 August 2012free access The hSSB1 orthologue Obfc2b is essential for skeletogenesis but dispensable for the DNA damage response in vivo Niklas Feldhahn Corresponding Author Niklas Feldhahn Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Search for more papers by this author Elisabetta Ferretti Elisabetta Ferretti Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Davide F Robbiani Davide F Robbiani Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Search for more papers by this author Elsa Callen Elsa Callen Laboratory of Genome Integrity, National Cancer Institute National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Stephanie Deroubaix Stephanie Deroubaix Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Search for more papers by this author Licia Selleri Licia Selleri Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Andre Nussenzweig Andre Nussenzweig Laboratory of Genome Integrity, National Cancer Institute National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Michel C Nussenzweig Michel C Nussenzweig Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Niklas Feldhahn Corresponding Author Niklas Feldhahn Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Search for more papers by this author Elisabetta Ferretti Elisabetta Ferretti Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Davide F Robbiani Davide F Robbiani Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Search for more papers by this author Elsa Callen Elsa Callen Laboratory of Genome Integrity, National Cancer Institute National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Stephanie Deroubaix Stephanie Deroubaix Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Search for more papers by this author Licia Selleri Licia Selleri Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Andre Nussenzweig Andre Nussenzweig Laboratory of Genome Integrity, National Cancer Institute National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Michel C Nussenzweig Michel C Nussenzweig Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Niklas Feldhahn 1, Elisabetta Ferretti2, Davide F Robbiani1, Elsa Callen3, Stephanie Deroubaix1, Licia Selleri2, Andre Nussenzweig3 and Michel C Nussenzweig1,4 1Laboratory of Molecular Immunology, Rockefeller University, New York, NY, USA 2Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA 3Laboratory of Genome Integrity, National Cancer Institute National Institutes of Health, Bethesda, MD, USA 4Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA *Corresponding author. Laboratory of Molecular Immunology, The Rockefeller University, 1230 York Avenue, Box 220, New York, NY 10065, USA. Tel.:+1 212 327 8098; Fax:+1 212 327 8370; E-mail: [email protected] The EMBO Journal (2012)31:4045-4056https://doi.org/10.1038/emboj.2012.247 Correction(s) for this article The hSSB1 orthologue Obfc2b is essential for skeletogenesis but dispensable for the DNA damage response in vivo15 January 2021 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human single-stranded DNA-binding protein 1 (hSSB1), encoded by OBFC2B, was recently characterized as an essential factor for the initiation of DNA damage checkpoints and the maintenance of genomic stability. Here, we report that loss of Obfc2b in mice results in perinatal lethality characterized by growth delay and skeletal abnormalities. These abnormalities are associated with accumulation of γH2ax, apoptosis and defective pre-cartilage condensation, which is essential for normal bone formation. However, deficiency of Obfc2b does not affect the initiation of DNA damage checkpoints, Atm activation, or the maintenance of genomic stability in B lymphocytes and primary fibroblasts. Loss of Obfc2b results in increased expression of its homologue Obfc2a (hSSB2). In contrast to Obfc2b deficiency, depletion of Obfc2a in fibroblasts results in impaired proliferation, accumulation of γH2ax and increased genomic instability. Thus, the hSSB1 orthologue Obfc2b has a unique function during embryogenesis limited to cell types that contribute to bone formation. While being dispensable in most other cell lineages, its absence leads to a compensatory increase in Obfc2a protein, a homologue required for the maintenance of genomic integrity. Introduction Nuclear DNA is normally double-stranded, but single-stranded DNA (ssDNA) is exposed during DNA replication, meiosis, transcription, and DNA double-strand break (DSB) repair. ssDNA, which is an obligate intermediate in these reactions, is more vulnerable to chemical and physical damage than double-stranded DNA (dsDNA). The increased vulnerability of ssDNA is alleviated in part by ssDNA-binding proteins that stabilize, protect and facilitate the repair of damaged ssDNA (reviewed by Mendez and Stillman, 2003). Underlining their importance, loss of function of the ssDNA-binding protein replication protein A (Rpa1) results in embryonic lethality in mice (Wang et al, 2005). Even heterozygous Rpa1 mutant mice show an increase in genomic instability and develop lymphoid tumours (Wang et al, 2005). Two additional ssDNA-binding proteins, hSSB1 (OBFC2B, NABP2 or SOSS-B1) and hSSB2 (OBFC2A, NABP1 or SOSS-B2), are also thought to be essential for recognition and repair of DNA damage (Richard et al, 2008, 2011a, 2011b; Huang et al, 2009; Li et al, 2009; Zhang et al, 2009). Similarly to RPA1, hSSB1 and hSSB2 form heterotrimeric complexes that are required for their recruitment to DSBs (Huang et al, 2009; Li et al, 2009; Skaar et al, 2009; Zhang et al, 2009). RNA interference (RNAi) experiments indicated that hSSB1 is essential to induce phosphorylation of ataxia telangiectasia mutated (ATM) kinase and its downstream targets in response to DNA damage. Moreover knockdown of hSSB1 is reported to abrogate irradiation-induced G1/S and G2/M cell-cycle arrest and result in genomic instability (Richard et al, 2008; Huang et al, 2009; Li et al, 2009; Zhang et al, 2009). In addition to repair and checkpoint functions, it has been proposed that hSSB1 is also required to produce ssDNA at sites of DSBs and that it does so by recruiting the MRN (MRE11/RAD50/NBS1) complex and the CtBP-interacting protein (CTIP) endonuclease (Richard et al, 2011a, 2011b). However, the role of hSSB1 in DNA repair has only been tested in RNAi knockdown experiments in cell lines. To study the role of the ssDNA-binding protein hSSB1 in vivo, we produced conditional knockout mice for the hSSB1 orthologue Obfc2b. We find that Obfc2b exhibits an essential, unique and cell type-specific role during embryogenesis. Germline deletion of Obfc2b results in increased replication-associated DNA damage and apoptosis in cell types that are essential for skeletal development and, hence, in severe skeletal defects and perinatal lethality. Furthermore, loss of Obfc2b results in a compensatory increase of its homologue Obfc2a (orthologue to hSSB2). Unexpectedly, these ssDNA-binding proteins are not required to initiate the DNA damage response to irradiation, but play an important tissue-specific role in the suppression of replication-associated DNA damage. Results Germline deletion of Obfc2b results in embryonic lethality Human ssDNA-binding protein 1 (hSSB1 or SOSS-B1) is encoded by the OBFC2B gene (oligonucleotide/oligosaccharide-binding fold containing 2B; Supplementary Figure 1A). To conditionally delete hSSB1, in mice, we introduced loxP sites flanking exons 1 and 2 of Obfc2b in mouse embryonic stem (ES) cells (Obfc2blox; Figure 1A and Supplementary Figure 1B). To generate mice carrying an Obfc2b knockout allele, Obfc2blox mice were bred with mice expressing the EIIACre transgene (Lakso et al, 1996; Supplementary Figure 1B and C). Cre-mediated loss of Obfc2b protein was confirmed by western blotting of B cells from CD19Cre; Obfc2blox/− mice (Supplementary Figure 1D and see below). Figure 1.Loss of Obfc2b results in embryonic lethality and developmental abnormalities. (A) Design of the conditional Obfc2b allele. Schematic of the murine Obfc2blox allele with integrated loxP sites before (upper panel), and after Cre-mediated disruption (lower panel) is shown. (B) Obfc2b deficiency results in embryonic lethality. Table of genotypes observed from Obfc2b+/− intercrosses are shown. (C) Obfc2b deficiency results in developmental abnormalities. Appearance of embryos at days E18.5 (left) and P0 (right) is shown. Bar marks size difference and arrows mark developmental abnormalities of the hindlimbs. (D) Obfc2b mRNA analysis in wild-type embryos at day E10.5 by in situ hybridization. Arrows indicate specific staining at the branchial arches (BAs), neural crest (NC), neural tube (NT), forelimbs (FL) and hindlimbs (HL) and somites (So) using the anti-sense probe. An Obfc2b sense probe was used as control (right). Download figure Download PowerPoint To determine whether Obfc2b is essential for mouse development, Obfc2b+/− mice were interbred. Out of 129 pups analysed at 0–2 weeks of age, we found no viable Obfc2b−/− mice even on the day of delivery (P0) (Figure 1B). However, Obfc2b−/− embryos were present at nearly Mendelian ratios as late as at embryonic day 18.5 (E18.5, Figure 1B). At this time, the homozygous mutant embryos appeared to be viable but exhibited significant growth delay, rudimentary hindlimbs (HL) and an abnormal skull (Figure 1C). We conclude that loss of Obfc2b results in developmental abnormalities during embryogenesis and perinatal death. To determine whether the developmental abnormalities in Obfc2b−/− mice result from a tissue-specific requirement of Obfc2b function during embryogenesis, we performed in situ hybridization for Obfc2b mRNA expression on wild-type E10.5 embryos. Obfc2b was expressed in several tissues that contribute to the development of skeletal structures (Figure 1D). These include the limb buds that organize the development of fore- and hindlimbs (FL, HL); the somites (So) which form in part the sclerotome and further the vertebrae and part of the skull; the branchial arches (BAs) that contribute to the development of the mandibles and the palate; and the prospective neural crest (NC) that can give rise to craniofacial mesenchyme and further form craniofacial cartilage and bones. In addition, Obfc2b mRNA expression seemed to be specific for the closing neural tube (NT) and different regions of the head (Figure 1D). We conclude that Obfc2b shows a tissue-specific expression pattern during normal embryogenesis. Obfc2b−/− embryos exhibit severe skeletal defects To characterize skeletal defects in more depth, we visualized cartilage and mineralized bone in E18.5 embryos (Figure 2). Obfc2b−/− embryos exhibited severe skeletal abnormalities affecting the rib cage, limbs and skull: the rib cage showed a general decrease in size and ossified rib segments were missing or rudimentary (Figure 2A). Since the lower rib cage serves as an anchor for the thoracic diaphragm, it is likely that the perinatal death of Obfc2b−/− embryos is caused by respiratory failure. Micro-computer tomography (MicroCT) analysis of dead Obfc2b−/− newborns (P0) confirmed the presence of a rudimentary rib cage and revealed that the bones were thinner and showed increased porosity (Supplementary Figure 2). Furthermore, the skull had a hypoplastic lower mandible (Figure 2B, left), the tympanic ring of the inner ear was rudimentary and the palate was cleft (Figure 2B, right). In the region of the forelimbs, the spine of the scapula and the deltoid tuberosity of the humerus were missing or rudimentary, respectively (Figure 2C, left). There was variable penetrance of skeletal abnormalities in the hindlimbs, which included missing digits (Figure 2C, right). MicroCT of the femurs revealed a significant decrease in bone volume (Supplementary Figure 2B–D). This pattern of skeletal defects is consistent with the pattern of Obfc2b expression in wild-type E10.5 embryos described above (Figure 1D). We conclude that Obfc2b deficiency results in multiple skeletal defects during embryogenesis. Figure 2.Skeletal abnormalities in Obfc2b−/− embryos. (A) Rib cage preparations of E18.5 embryos visualized by Alcian blue and Alizarin red staining indicating cartilage (blue) and mineralized/ossified tissue (red). (B) Skull preparations: bars mark size differences in the lower mandible (left) and arrows mark the rudimentary tympanic ring and the cleft palate in skulls from Obfc2b−/− embryos (middle and right). (C) Preparations of the forelimbs (left panels) and hindlimbs from wild-type and Obfc2b−/− embryos (right panels). Download figure Download PowerPoint Osteoblasts, chondrocytes and osteoblasts in Obfc2b−/− mice To determine whether skeletal defects in Obfc2b−/− mice arise as a consequence of aberrant differentiation or function of bone forming cells, we isolated osteoblasts, chondrocytes and osteoclasts from wild-type and Obfc2b−/− mice. Osteoblasts were isolated from the calvaria of the skull from E18.5 embryos; chondrocytes were isolated from the sternum of the ribcage. Osteoclasts were generated from bone marrow cells by stimulation with RANK-L and M-CSF for 5 days in culture and osteoclast identity was verified by Tartrate-resistant Acidic Phosphatase (TRAP) staining (Supplementary Figure 3A). Isolated cells were then subjected to gene expression analysis using gene arrays. Expression of osteoblast-, chondrocyte- and osteoclast-specific genes confirmed the identity of the isolated cell subsets (Supplementary Figure 3B). Comparison of gene arrays from wild-type and Obfc2b−/− cells showed that Obfc2b was expressed in wild-type but not in Obfc2b−/− cells. However, wild-type and Obfc2b−/− cells were otherwise indistinguishable (Supplementary Figure 3C). We conclude that deficiency of Obfc2b does not lead to significant changes in osteoblast, chondrocyte or osteoclast gene expression. Skeletal defects are associated with increased apoptosis Since the human Obfc2b orthologue hSSB1 has been implicated in DNA repair and DNA repair deficiencies can result in apoptosis during embryogenesis (Gao et al, 1998), we asked whether the skeletal defects in Obfc2b−/− embryos were associated with increased apoptosis. To detect apoptotic cells, we performed terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) on tissue sections from E10.5, E12.5 and E16.5 embryos. Whereas E10.5 and E16.5 Obfc2b−/− embryos displayed no notable abnormalities (Supplementary Figure 4A and B), E12.5 Obfc2b−/− embryos showed significantly increased numbers of apoptotic cells in the developing ribs, hindlimb (HL) bud and branchial arches (BAs) (Figure 3A; Supplementary Figure 4C). This pattern of TUNEL staining is consistent with the majority of the described skeletal defects at E18.5 (see Figure 2). Figure 3.Increased skeletal apoptosis in Obfc2b−/− embryos. (A) Obfc2b deficiency is associated with increased apoptosis at E12.5. Terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL) staining of representative tissue sections of E12.5 embryos. Arrows indicate regions of developing branchial arches (BAs), developing ribs and developing hindlimbs (HL). (B) P53 loss partially rescues skeletal abnormalities in Obfc2b−/− embryos. Alcian blue and Alizarin red stained skeletal preparations from P0 Obfc2b−/−;p53+/+, Obfc2b−/−;p53−/−, Obfc2b+/+;p53−/− and Obfc2b+/+;p53+/+ embryos as in Figure 2. Preparations of the rib cage (upper panels), and of the scapula of the forelimb (lower left) and the autopod of the hindlimb (lower right) are shown. (C) Western blot analysis of hind- and forelimbs from E12.5 embryos for phospho-p53 (serine 15), γH2ax, Obfc2b and Lamin b1. (D) In situ hybridization of whole embryos (E12.5) for Sox9 mRNA. Upper panels: images of whole embryos are shown including a magnification of the developing scapula. White arrows indicate aberrant Sox9 staining at the scapula and at the hindlimb. Lower panels: Sox9 staining of forelimbs (left) and hindlimbs (right). Download figure Download PowerPoint To determine whether the skeletal defects in Obfc2b−/− embryos can be rescued by loss of p53, we interbred the two mutant mouse strains to generate Obfc2b−/−; p53−/− mice. In contrast to Obfc2b−/− newborns that were grossly abnormal and not viable, Obfc2b−/−; p53−/− mice appeared normal, were viable at birth, and survived up to 24 h. Skeletal preparations from Obfc2b−/−; p53−/− mice at P0 showed partial rescue of the thoracic rib cage phenotype (Figure 3B, up), and of the forelimb and hindlimb defects (Figure 3B, low). However, the defects in the skull including the cleft palate were not ameliorated and likely account for the perinatal lethality of the Obfc2b/p53 double knockout pups due to the inability to feed. We conclude that most skeletal defects in Obfc2b−/− embryos are associated with increased apoptosis and, accordingly, can be partially rescued by loss of p53. Skeletal defects are associated with increased genomic instability at E12.5 To investigate whether increased apoptosis in Obfc2b−/− embryos results from increased genomic instability, we analysed forelimbs and hindlimbs from E12.5 embryos for γH2ax accumulation and p53 phosphorylation by western blotting because both are induced by and serve as markers for DNA damage (Figure 3C). We found that hindlimbs from Obfc2b−/− embryos showed increased γH2ax accumulation and p53 phosphorylation at serine 15 (Figure 3C). In contrast, there was only a minimal increase of γH2ax accumulation and p53 phosphorylation in the forelimbs of Obfc2b−/− embryos, which show only minor skeletal defects. We conclude that the skeletal defects in the limbs of Obfc2b−/− embryos are associated with and likely to result from increased genomic instability and apoptosis at E12.5. Defective pre-cartilage condensation at E12.5 in Obfc2b−/− embryos Pre-cartilage mesenchymal condensation precedes the development of chondrocytes and osteoblasts and is essential for the development of skeletal structures (reviewed in Hall and Miyake, 2000 and Kronenberg, 2003). Apoptosis is likely to antagonize pre-cartilage condensation, because defects in skeletogenesis are often associated with increased apoptosis (Akiyama et al, 2002; Cheung et al, 2005; Shim et al, 2010). Conversely, the transcription factor Sox9, which is specifically expressed during condensation in forelimbs and hindlimbs (Wright et al, 1995), is thought to suppress apoptosis (Akiyama et al, 2002). To analyse if increased apoptosis in Obfc2b−/− embryos is associated with defective pre-cartilage condensation we performed in situ hybridization for Sox9 on whole embryos at E12.5 (Figure 3D). In agreement with the presence of a minimal skeletal defect in the forelimb of Obfc2b−/− embryos, pre-cartilage condensation appeared to be mostly normal in the developing forelimbs of Obfc2b−/− embryos (Figure 3D, top and lower left). In contrast, the hindlimbs, which show a high degree of apoptosis and skeletal defects, showed defective pre-cartilage condensation, as did the developing scapula in the forelimb (Figure 3D, up and lower right). We conclude that increased genomic instability and apoptosis in Obfc2b−/− embryos is associated with defective pre-cartilage mesenchymal condensation. Obfc2b is dispensable for the DNA damage response in B lymphocytes and MEFs Lymphocytes are especially sensitive to defects in the DNA damage response because they undergo programmed DNA damage during V(D)J recombination, and immunoglobulin class-switch recombination (CSR in B cells). As a result, lymphocytes development and function is abnormal in mice or humans that are mutant in any of a number of different factors that contribute to the recognition and repair of DNA damage (reviewed in Rooney et al, 2004; Dudley et al, 2005 and Jankovic et al, 2007). The Obfc2b orthologue hSSB1 has been postulated to be required for the recognition of DNA damage and its repair by the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways (Richard et al, 2008, 2011a, 2011b). To circumvent embryonic lethality and analyse Obfc2b function in B cells, we deleted it specifically by combining the conditional allele with a lineage-specific Cre transgene (CD19Cre;Obfc2blox/− mice). Despite undetectable levels of Obfc2b protein in CD19Cre;Obfc2blox/− B lymphocytes, B cell development in the bone marrow was indistinguishable from control mice (Figure 4A). Likewise, T cell development was indistinguishable in lethally irradiated mice reconstituted with fetal liver cells from wild-type or Obfc2b−/− embryos (Figure 4B). Moreover, immunoglobulin CSR in stimulated B cells was unaffected by loss of Obfc2b (Figure 4C). Since CSR and V(D)J recombination require efficient recognition of DNA damage and its repair by the NHEJ pathway we conclude that Obfc2b is not required for DNA damage sensing or its repair by NHEJ in lymphocytes. Figure 4.DNA damage response in Obfc2b−/− B cells. (A, B) Normal B and T cell development in Obfc2b−/− mice. (A) Flow cytometry analysis of control (CD19Cre;Obfc2b+/+) and conditional Obfc2b knockout (CD19Cre;Obfc2blox/−) mice. B cell subsets in the bone marrow were identified using the antibodies indicated. (B) Normal T cell development in Obfc2b−/− mice. Thymocytes from lethally irradiated mice reconstituted with fetal liver cells from Obfc2b+/+ or Obfc2b−/− embryos. Analysis was performed >2 months after reconstitution. (C) Normal class switch-recombination (CSR) in Obfc2b−/− B cells. Bar diagram shows mean values of IgG surface expression on conditional Obfc2b knockout (CD19Cre;Obfc2blox/−) and control B cells (CD19Cre/+;Obfc2b+/+ and CD19Cre;Obfc2blox/+) after 4 days of proliferation in vitro, as determined by flow cytometry. IgG1 expression was induced by LPS, IL-4 and RP105 (*) or by LPS and IL-4, IgG3 expression was induced by LPS alone. (D) Normal PARPi sensitivity in Obfc2b−/− B cells. B cells from Obfc2b wild-type mice (CD19Cre;Obfc2b+/+), conditional Obfc2b heterozygous mice (CD19Cre;Obfc2blox/+), conditional Obfc2b knockout mice (CD19Cre;Obfc2blox/−), Atm−/− mice or wild-type B cells treated with 2.5 μM Ku55933 (ATMi) were stained with carboxyfluorescein succinimidyl ester (CFSE) and analysed by flow cytometry after 4 days in culture with LPS and IL-4. Cells were cultured with or without 1 μM Ku58948 (PARPi). Bar diagram shows mean values of five individual experiments. Data observed for Atm−/− B cells and wild-type B cells treated with ATMi have been pooled. For gating of CFSE-positive cells, see Supplementary Figure 5. (E) Normal radiosensivity in Obfc2b−/− B cells. Metaphase analysis of proliferating B cells from control (CD19Cre;Obfc2b+/+ and CD19Cre;Obfc2blox/+) and conditional Obfc2b knockout mice (CD19Cre;Obfc2blox/−) after irradiation with 6 Gy (20 h recovery). Bar diagram shows the mean values of eight pairs of mice analysed in five individual experiments. (F) Obfc2b−/− B cells exhibit a slight increase of c-myc/Igh translocations. PCR analysis of c-myc/Igh translocations in B cells from control (CD19Cre;Obfc2b+/+ and CD19Cre;Obfc2blox/+) and conditional Obfc2b knockout mice (CD19Cre;Obfc2blox/−). B cells were cultured for 4 days with LPS and IL-4 previous to analysis. Bar diagram shows the mean of three and four independent experiments for derivative chromosomes 15 and 12, respectively. Data for both derivative chromosomes has been pooled. Download figure Download PowerPoint Poly (ADP-ribose) polymerase (PARP) is required for the detection of single-strand DNA breaks. Inhibiting this enzyme with Ku58948 (PARPi) destabilizes the genome by increasing the number of ssDNA breaks, many of which develop into DSBs that are repaired by HR (Bryant et al, 2005; Jackson and Bartek, 2009). Cells defective in HR as well as cells treated with the ATM inhibitor Ku55933 (ATMi) are especially sensitive to PARPi treatment and show reduced proliferation in the presence of PARPi (Bunting et al, 2010). To determine whether Obfc2b is required for HR, we measured B cell proliferation in the presence or absence of PARPi (Figure 4D). Proliferation was measured by cell division-associated decrease of cytoplasmic staining with carboxyfluorescein succinimidyl ester (CFSE). Cells delayed in proliferation remain CFSE positive after 4 days. Atm−/− B cells or wild-type B cells treated with the ATM inhibitor Ku55933 (ATMi) were used as positive controls, and showed a significant increase of cells with delayed proliferation upon PARPi treatment (CFSE-positive cells; Figure 4D and Supplementary Figure 5). However, there was no measurable effect upon loss of Obfc2b. We conclude that Obfc2b is not required for DNA repair by HR in proliferating B lymphocytes. To further test if Obfc2b is required for the recognition and repair of DNA damage, we irradiated proliferating B cells and analysed metaphases for unrepaired genomic aberrations by fluorescence in situ hybridization (FISH). We found only a small and statistically insignificant increase in genomic aberrations in metaphases from activated Obfc2b−/− B cells after ionizing irradiation (IR; Figure 4E). We next analysed the frequency of c-myc/Igh translocations in proliferating B cells, which are a byproduct of CSR. Defects in recognition and repair of DNA damage typically result in a significant increase in such translocations (Ramiro et al, 2006). The frequency of c-myc/Igh translocations in proliferating Obfc2b deficient B cells was slightly elevated compared to Obfc2b proficient cells (Figure 4F). Nevertheless, the translocation frequencies observed fall within the range of what is typically reported for wild-type B cells (Ramiro et al, 2004, 2006). We conclude that the hSSB1 orthologue Obfc2b is not required to maintain genomic stability in dividing B lymphocytes. It has been suggested that hSSB1 is required for activation of the G1/S and the G2/M DNA damage checkpoint upon irradiation (Richard et al, 2008). To determine whether the hSSB1 orthologue Obfc2b is required for the initiation of DNA damage checkpoints in vivo, we analysed B cells from CD19Cre;Obfc2blox/− mice for cell-cycle arrest in response to irradiation. Cells entering mitosis undergo histone 3 phosphorylation at serine 10. Accordingly, histone 3 phosphorylation is abrogated in response to G2/M checkpoint activation. As expected, inhibition of Atm kinase activity using Ku55933 (ATMi) resulted in a reduced activation of the G2/M checkpoint in response to irradiation (Fernandez-Capetillo et al, 2002; Figure 5A). However, there was no measurable effect of Obfc2b ablation compared to wild-type cells (Figure 5A; Supplementary Figure 6A). Similarly, loss of Obfc2b did not affect the G1/S checkpoint as measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation (Figure 5B; Supplementary Figure 6B): Upon IR, Obfc2b-deficient cells (KO) were indistinguishable from wild-type controls in terms of cell-cycle distribution (Figure 5B). Consistent with the absence of cell-cycle checkpoint defects in response to IR, Western blot analysis showed no alterations in IR-induced phosphorylation of Atm, Chk1 (a substrate of Atr) or p53 in the absence of Obfc2b (Figure 5C; Supplementary Figure 6C). Similar results were also obtained with Obfc2b−/− primary murine embryonic fibroblasts (MEFs; Supplementary Figure 6D and E). Thus, Obfc2b is dispensable for Atm/Atr activation and the initiation of DNA damage checkpoints in primary B lymphocytes and embryonic fibroblasts. Figure 5.DNA damage checkpoint analysis. (A) Normal G2/M checkpoint in Obfc2b−/− B cells. Proliferating B cells from wild-type (WT, CD19Cre;Obfc2b+/+) and conditional knockout (KO, CD19Cre;Obfc2blox/−) mice were analysed for histone 3 (serine 10) phosphorylation before and after irradiation (1 h recovery). As a control, wild-type (CD19Cre/+;Obfc2b+/+) B cells treated with 2.5 μM Ku55933 (ATMi) were analysed. The graph represents the results from three pairs of mice and two independent experiments. (B) Normal G1/S checkpoint in Obfc2b−/− B cells. Proliferating B cells from wild-type (WT, CD19Cre;Obfc2b+/+) and conditional knockout (KO, CD19Cre;Obfc2blox/−) mice were pulsed with BrdU and subjected to cell-cycle analysis. Cells were either mock treated or irradiated with 6 Gy, and allowed to recover for 0.5–24 h. Cells in S phase, G1 and G2 are plotted as individual graphs. Graphs represent the results from three pairs of mice and two independent experiments. (C) Normal IR-induced phosphorylations in Obfc2b−/− B cells. Western
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