Haploinsufficiency of Parp1 accelerates Brca1-associated centrosome amplification, telomere shortening, genetic instability, apoptosis, and embryonic lethality

The breast tumor associated gene-1 (BRCA1) and poly(ADP-ribose) polymerase-1 (PARP1) are both involved in DNA-damage response and DNA-damage repair. Recent investigations have suggested that inhibition of PARP1 represents a promising chemopreventive/therapeutic approach for specifically treating BRCA1- and BRCA2-associated breast cancer. However, studies in mouse models reveal that Parp1-null mutation results in genetic instability and mammary tumor formation, casting significant doubt on the safety of PARP1 inhibition as a therapy for the breast cancer. To study the genetic interactions between Brca1 and Parp1, we interbred mice carrying a heterozygous deletion of full-length Brca1 (Brca1(+/Delta11)) with Parp1-null mice. We show that Brca1(Delta11/Delta11);Parp1(-/-) embryos die before embryonic (E) day 6.5, whereas Brca1(Delta11/Delta11) embryos die after E12.5, indicating that absence of Parp1 dramatically accelerates lethality caused by Brca1 deficiency. Surprisingly, haploinsufficiency of Parp1 in Brca1(Delta11/Delta11) embryos induces a severe chromosome aberrations, centrosome amplification, and telomere dysfunction, leading to apoptosis and accelerated embryonic lethality. Notably, telomere shortening in Brca1(Delta11/Delta11);Parp1(+/-) MEFs was correlated with decreased expression of Ku70, which plays an important role in telomere maintenance. Thus, haploid loss of Parp1 is sufficient to induce lethality of Brca1-deficient cells, suggesting that partial inhibition of PARP1 may represent a practical chemopreventive/therapeutic approach for BRCA1-associated breast cancer.     

Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice

Breast tumor suppressor gene 1 (BRCA1) plays an essential role in maintaining genomic integrity. Here we show that mouse Brca1 is required for DNA-damage repair and crossing-over during spermatogenesis. Male Brca1(Delta11/Delta11)p53(+/-) mice that carried a homozygous deletion of Brca1 exon 11 and a p53 heterozygous mutation had significantly reduced testicular size and no spermatozoa in their seminiferous tubules. During spermatogenesis, homologous chromosomes from the mutant mice synapsed and advanced to the pachytene stage but failed to progress to the diplotene stage. Our analyses revealed that the Brca1 mutation affected cellular localization of several DNA damage-repair proteins. This included prolonged association of gammaH2AX with sites of DNA damage, reduced sex body formation, diminished Rad51 foci and absence of Mlh1 foci in the pachytene stage. Consequently, chromosomes from mutant mice did not form chiasmata, a point that connects exchanging homologous chromosomes. Brca1-mutant spermatocytes also exhibited decreased RNA expression levels of several genes that are involved in DNA-damage repair, including RuvB-like DNA helicase, XPB, p62 and TFIID. Of note, the premature termination of spermatogenesis at the pachytene stage was accompanied by increased apoptosis by both p53-dependent and p53-independent mechanisms. Thus, our study revealed an essential role of Brca1 in DNA-damage repair and crossing-over of homologous chromosomes during spermatogenesis.     

Chk2 suppresses the oncogenic potential of DNA replication-associated DNA damage

The Mre11 complex (Mre11, Rad50, and Nbs1) and Chk2 have been implicated in the DNA-damage response, an inducible process required for the suppression of malignancy. The Mre11 complex is predominantly required for repair and checkpoint activation in S phase, whereas Chk2 governs apoptosis. We examined the relationship between the Mre11 complex and Chk2 in the DNA-damage response via the establishment of Nbs1(DeltaB/DeltaB) Chk2(-/-) and Mre11(ATLD1/ATLD1) Chk2(-/-) mice. Chk2 deficiency did not modify the checkpoint defects or chromosomal instability of Mre11 complex mutants; however, the double-mutant mice exhibited synergistic defects in DNA-damage-induced p53 regulation and apoptosis. Nbs1(DeltaB/DeltaB) Chk2(-/-) and Mre11(ATLD1/ATLD1) Chk2(-/-) mice were also predisposed to tumors. In contrast, DNA-PKcs-deficient mice, in which G1-specific chromosome breaks are present, did not exhibit synergy with Chk2(-/-) mutants. These data suggest that Chk2 suppresses the oncogenic potential of DNA damage arising during S and G2 phases of the cell cycle.     

Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.     

BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.     

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

BRCA2 is required for neurogenesis and suppression of medulloblastoma

Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2(LoxP/LoxP);Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2(LoxP/LoxP);Nestin-cre mice showed extensive gammaH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors.     

Loss of Bard1, the heterodimeric partner of the Brca1 tumor suppressor,results in early embryonic lethality and chromosomal instability

The BRCA1 tumor suppressor has been implicated in many cellular pathways, but the mechanisms by which it suppresses tumor formation are not fully understood. In vivo BRCA1 forms a heterodimeric complex with the related BARD1 protein, and its enzymatic activity as a ubiquitin ligase is largely dependent upon its interaction with BARD1. To explore the genetic relationship between BRCA1 and BARD1, we have examined the phenotype of Bard1-null mice. These mice become developmentally retarded and die between embryonic day 7.5 (E7.5) and E8.5. Embryonic lethality results from a severe impairment of cell proliferation that is not accompanied by increased apoptosis. In the absence of p53, the developmental defects associated with Bard1 deficiency are partly ameliorated, and the lethality of Bard1; p53-nullizygous mice is delayed until E9.5. This result, together with the increased chromosomal aneuploidy of Bard1 mutant cells, indicates a role for Bard1 in maintaining genomic stability. The striking similarities between the phenotypes of Bard1-null, Brca1-null, and double Bard1; Brca1-null mice provide strong genetic evidence that the developmental functions of Brca1 and Bard1 are mediated by the Brca1/Bard1 heterodimer.     

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Brca1-Deficient Murine Mammary Epithelial Cells have Increased Sensitivity to CDDP and MMS

In this report we describe the isolation of an isogenic pair of Brca1+/+ and Brca1-/-murine mammary epithelial cells (MMECs). These cells were isolated from Brca1conditional knock out mice which contained loxP sites flanking exon 11 of the Brca1gene (Brca1fl/fl) and then immortalized by infection with HPV-16E6 retrovirus to degradep53 protein. Brca1-/- MMECs were generated by deletion of exon 11 followingtransduction of Brca1fl/fl MMECs with a retroviral vector expressing Cre recombinase.Brca1-deficiency rendered MMECs sensitive to cis-platinum (II) diamine dichloride(CDDP) and methylmethane sulfonate (MMS). The Brca1+/+ and Brca1-/- MMECS is theonly known pair of isogenic mammary epithelial cell lines. The understanding of themechanisms of the CDDP sensitivity of the BRCA1-deficient mammary epithelial cellswould be very important in understanding how BRCA1-deficiency plays out in tissuespecific breast cancer chemotherapy. These studies support the role of BRCA1 in theCDDP-induced and MMS-induced DNA damage and repair by p53-independentpathways.     

p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.     

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a targeted mutation of the Chk2 phosphorylation site in Brca1

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.     

In vivo role of checkpoint kinase 2 in signaling telomere dysfunction

Checkpoint kinase 2 (CHK2) is a downstream effector of the DNA damage response (DDR). Dysfunctional telomeres, either owing to critical shortening or disruption of the shelterin complex, activate a DDR, which eventually results in cell cycle arrest, senescence and/or apoptosis. Successive generations of telomerase‐deficient (Terc) mice show accelerated aging and shorter lifespan due to tissue atrophy and impaired organ regeneration associated to progressive telomere shortening. In contrast, mice deficient for the shelterin component TRF1 in stratified epithelia show a rapid and massive induction of DDR, leading to perinatal lethality and severe skin defects. In both mouse models, p53 deficiency can rescue survival. Here, we set to address the role of CHK2 in signaling telomere dysfunction in both mouse models. To this end, we generated mice doubly deficient for Chk2 and either Terc (Chk2^?/? Terc^?/?) or Trf1 (Trf1^Δ/Δ K5Cre Chk2^?/?). We show that Chk2 deletion improves Terc‐associated phenotypes, including lifespan and age‐associated pathologies. Similarly, Chk2 deficiency partially rescues perinatal mortality and attenuates degenerative pathologies of Trf1^Δ/Δ K5Cre mice. In both cases, we show that the effects are mediated by a significant attenuation of p53/p21 signaling pathway. Our results represent the first demonstration of a role for CHK2 in the in vivo signaling of dysfunctional telomeres.     

Nbs1‐mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis

ObjectivesDNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood‐derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well‐defined.Materials and methodsMice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1‐p53 signaling in HSCs and haematopoietic progenitors.ResultsNbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm‐Chk2‐p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality.ConclusionsOur study discloses that DNA double‐strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.     

Tumorigenesis as a consequence of genetic instability in Brca1 mutant mice

Germline mutations in Brca1 are responsible for most cases of familial breast and ovarian cancers, but somatic mutations in the gene are rarely detected in sporadic tumors. Moreover, mouse embryos carrying Brca1-null mutations or homozygous deletions of Brca1 exon 11 of (Brca1Delta11/Delta11) die during gestation due to proliferation defects, raising questions about the mechanisms by which Brca1 represses tumor formation. Molecular analysis reveals that these Brca1 mutations cause hypersensitivity to gamma-irradiation and chromosomal abnormalities in embryos and embryonic fibroblast cells (MEFs). Notably, Brca1Delta11/Delta11 MEFs maintain an intact G1-S checkpoint, but are defective in G2-M checkpoint control. They also contain multiple, functional centrosomes, which lead to unequal chromosome segregation and aneuploidy. These data uncover an essential role for Brca1 in maintaining genetic stability through regulation of centrosome duplication and G2-M checkpoint, and provide a molecular basis for its role in tumorigenesis. Finally, we show that conditional mutation of Brca1 in mammary epithelium causes increased apoptosis and abnormal ductal development. Mammary tumor formation in mutant mice occurs after long latency and is associated with p53 mutations. These results are consistent with a model that Brca1 acts as a caretaker gene, whose absence does not directly initiate tumorigenesis, instead, causes genetic instability, which triggers further alterations and ultimately leads to tumor formation.     

An essential role for REV3 in mammalian cell survival: absence of REV3 induces p53-independent embryonic death

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.