RESUMO
Germline mutations of homologous-recombination (HR) genes are among the top contributors to medulloblastomas. A significant portion of human medulloblastomas exhibited genomic signatures of HR defects. We asked whether ablation of Brca2, Palb2, and their related Brca1 and Bccip genes, in the mouse brain can differentially initiate medulloblastomas. We established conditional knockout mouse models of these HR-genes, and a conditional knockdown of Bccip (shBccip-KD). Deletion of any of these genes led to microcephaly and neurological defects, with Brca1- and Bccip- producing the worst. Trp53 co-deletion significantly rescued the microcephaly with Brca1, Palb2, and Brca2 deficiency, but it exhibited limited impact on Bccip- mice. For the first time, inactivation of either Brca1 or Palb2 with Trp53 was found to induce medulloblastomas. Bccip/Trp53 deletions failed, despite that shBccip-CKD was highly penetrative, to induce medulloblastomas. The tumors displayed diverse immunohistochemical features and chromosome copy number variation. While there were widespread upregulations of cell proliferative pathways, most of the tumors expressed biomarkers of the Sonic Hedgehog subgroup. The MBs developed from Brca1-, Palb2-, and Brca2- mice were highly sensitive to a PARP inhibitor, but not the ones from shBccip-CKD mice. Our models recapitulate the spontaneous medulloblastoma development with high penetrance and narrow time-window, providing ideal platforms to test therapeutic agents with the ability to differentiate HR defective and proficient tumors.
RESUMO
The mammalian non-homologous end joining (NHEJ) is required for V(D)J recombination as well as coping with exogenously induced DNA double strand breaks (DSBs). Initiated by the binding of KU70/KU80 (KU) dimer to DNA ends and the subsequent recruitment of the DNA- dependent protein kinase catalytic subunit (DNA-PKcs), NHEJ plays a key role in DNA repair. While there has been significant structural understandings of how KU70 participates in NHEJ, the specific function of its highly conserved C-terminal SAP domain remains elusive. In this study, we developed a novel mouse model by deleting the SAP domain but preserving the KU70 nuclear localization and its dimerization ability with KU80. We found that the KU70 SAP deletion did not affect the V(D)J recombination or animal development but significantly impaired the animals and cells in repairing exogenously induced DSBs. We further showed an inability of KU70-ΔSAP cells to retain the DNA Ligase IV (LIG4) and other NHEJ co-factors on chromatin, and a spreading pattern of DSB marker γH2AX in KU70-ΔSAP cells after DNA damage. Our findings suggest that a specific inhibition of the SAP function may offer an opportunity to modulate cell sensitivity to therapeutic DSB-inducing agents without interfering with the developmental function of KU70. KeyPoints: Generation of a novel transgenic mouse line lacking the C-terminal conserved KU70-SAP domainKU70-SAP defends against exogenous DSBs, but unessential for development and V(D)J recombinationKU70-SAP aids in recruiting and retaining NHEJ components, such as LIG4, to DSB sites.
RESUMO
Sex chromosomes are subject to sex-specific selective evolutionary forces. One model predicts that genes with sex-biased expression should be enriched on the X chromosome. In agreement with Rice's hypothesis, spermatogonial genes are over-represented on the X chromosome of mice and sex- and reproduction-related genes are over-represented on the human X chromosome. Male-biased genes are under-represented on the X chromosome in worms and flies, however. Here we show that mouse spermatogenesis genes are relatively under-represented on the X chromosome and female-biased genes are enriched on it. We used Spo11(-/-) mice blocked in spermatogenesis early in meiosis to evaluate the temporal pattern of gene expression in sperm development. Genes expressed before the Spo11 block are enriched on the X chromosome, whereas those expressed later in spermatogenesis are depleted. Inactivation of the X chromosome in male meiosis may be a universal driving force for X-chromosome demasculinization.
Assuntos
Meiose/genética , Cromossomo X/genética , Animais , Mecanismo Genético de Compensação de Dose , Endodesoxirribonucleases , Esterases/deficiência , Esterases/genética , Feminino , Expressão Gênica , Genoma , Masculino , Camundongos , Camundongos Knockout , Modelos Genéticos , Gravidez , Espermatogênese/genéticaRESUMO
BCL-2-like protein 1 (BCL2L1) is a key component of cell survival and death mechanisms. Its dysregulation and altered ratio of splicing variants associate with pathologies. However, isoform-specific loss-of-function analysis of BCL2L1 remains unexplored. Here we show the functional impact of genetically inhibiting Bcl-x short-isoform (Bcl-xS) in vivo. Bcl-xS is expressed in most tissues with predominant expression in the spleen and blood cells in mice. Bcl-xS knockout (KO) mice show no overt abnormality until 3 months of age. Thereafter, KO mice develop cardiac hypertrophy with contractile dysfunction and splenomegaly by 6 months. Cardiac fibrosis significantly increases in KO, but the frequency of apoptosis is indistinguishable despite cardiomyopathy. The Akt/mTOR and JNK/cJun signaling are upregulated in male KO heart, and the JNK/cJun is activated with increased Bax expression in KO spleen. These results suggest that Bcl-xS may be dispensable for development but is essential for maintaining the homeostasis of multiple organs.
RESUMO
The mammalian KU70 is a pleiotropic protein functioning in DNA repair and cytoplasmic suppression of apoptosis. We report a regulatory mechanism by which KU70's cytoplasmic function is enabled due to a methylation at K570 of KU70 by SET-domain-containing protein 4 (SETD4). While SETD4 silencing reduces the level of methylated KU70, over-expression of SETD4 enhances methylation of KU70. Mutations of Y272 and Y284 of SETD4 abrogate methylation of KU70. Although SETD4 is predominantly a nuclear protein, the methylated KU70 is enriched in the cytoplasm. SETD4 knockdown enhances staurosporine (STS)-induced apoptosis and cell killing. Over-expression of the wild-type (WT) SETD4, but not the SETD4-Y272/Y284F mutant, suppresses STS-induced apoptosis. The KU70-K570R (mouse Ku70-K568R) mutation dampens the anti-apoptosis activity of KU70. Our study identifies KU70 as a non-histone substrate of SETD4, discovers a post-translational modification of KU70, and uncovers a role for SETD4 and KU70-K570 methylation in the suppression of apoptosis.
Assuntos
Apoptose , Reparo do DNA , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Apoptose/genética , Citoplasma/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Mamíferos/metabolismo , Metilação , Metiltransferases , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.
Assuntos
Proteínas de Transporte/genética , Dano ao DNA/genética , Fase G2/genética , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular , Mitose/genética , Fosfoproteínas , Proteínas Serina-Treonina Quinases , Animais , Linhagem Celular , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Fase G2/fisiologia , Regulação da Expressão Gênica , Camundongos , Mitose/fisiologia , Fosforilação , Proteínas Quinases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
RAD51 paralogs are key components of the homologous recombination (HR) machinery. Mouse mutants have been reported for four of the canonical RAD51 paralogs, and each of these mutants exhibits embryonic lethality, although at different gestational stages. However, the phenotype of mice deficient in the fifth RAD51 paralog, XRCC3, has not been reported. Here we report that Xrcc3 knockout mice exhibit midgestational lethality, with mild phenotypes beginning at about E8.25 but severe developmental abnormalities evident by E9.0-9.5. The most obvious phenotypes are small size and a failure of the embryo to turn to a fetal position. A knockin mutation at a key ATPase residue in the Walker A box results in embryonic lethality at a similar stage. Death of knockout mice can be delayed a few days for some embryos by homozygous or heterozygous Trp53 mutation, in keeping with an important role for XRCC3 in promoting genome integrity. Given that XRCC3 is a unique member of one of two RAD51 paralog complexes with RAD51C, these results demonstrate that both RAD51 paralog complexes are required for mouse development.
Assuntos
Proteínas de Ligação a DNA , Recombinação Homóloga , Rad51 Recombinase , Adenosina Trifosfatases/genética , Animais , Proteínas de Ligação a DNA/genética , Feminino , Camundongos , Gravidez , Rad51 Recombinase/genéticaRESUMO
The S. cerevisiae Hop2 protein and its fission yeast homolog Meu13 are required for proper homologous chromosome pairing and recombination during meiosis. The mechanism of this requirement is, however, not understood. The previous studies in Saccharomyces suggested that Hop2 is a guardian of meiotic chromosome synapsis with the ability to prevent or resolve deleterious associations between nonhomologous chromosomes. We have generated a Hop2 knockout mouse that shows profound meiotic defects with a distinct and novel phenotype. Hop2(-/-) spermatocytes arrest at the stage of pachytene-like chromosome condensation. Axial elements are fully developed, but synapsis of any kind is very limited. Immunofluorescence analysis of meiotic chromosome spreads indicates that while meiotic double-stranded breaks are formed and processed in the Hop2 knockout, they fail to be repaired. In aggregate, the Hop2 phenotype is consistent with a direct role for the mouse Hop2 protein in promoting homologous chromosome synapsis.
Assuntos
Meiose/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermatócitos/fisiologia , Animais , Pareamento Cromossômico/fisiologia , Reparo do DNA/fisiologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Recombinação Genética/fisiologia , Espermatogênese/fisiologiaRESUMO
During meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. The molecular basis and biological function of both sex body formation and meiotic sex chromosome inactivation (MSCI) are unknown. A phosphorylated form of H2AX, a histone H2A variant implicated in DNA repair, accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. Here we show that the X and Y chromosomes of histone H2AX-deficient spermatocytes fail to condense to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.
Assuntos
Cromatina/metabolismo , Inativação Gênica/fisiologia , Histonas/deficiência , Meiose/genética , Cromossomos Sexuais/metabolismo , Espermatócitos/metabolismo , Testículo/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Rad51 Recombinase , Cromatina Sexual/genética , Cromatina Sexual/metabolismo , Aberrações dos Cromossomos Sexuais , Cromossomos Sexuais/genética , Espermatócitos/patologia , Testículo/anormalidades , Testículo/patologiaRESUMO
The DNA-damage repair pathway homologous recombination (HR) requires factors that promote the activity of strand-exchange protein RAD51 and its meiosis-specific homolog DMC1. Here we show that the Shu complex SWS1-SWSAP1, a candidate for one such HR regulator, is dispensable for mouse viability but essential for male and female fertility, promoting the assembly of RAD51 and DMC1 on early meiotic HR intermediates. Only a fraction of mutant meiocytes progress to form crossovers, which are crucial for chromosome segregation, demonstrating crossover homeostasis. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility in females without rescuing crossover numbers. Concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Swsap1 mutant spermatocytes, suggesting an overlapping role with the Shu complex during meiotic HR. These results demonstrate an essential role for SWS1-SWSAP1 in meiotic progression and emphasize the complex interplay of factors that ensure recombinase function.
Assuntos
Meiose , Recombinação Genética , Animais , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Pareamento Cromossômico , Troca Genética , DNA/metabolismo , Feminino , Infertilidade/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Fosfato , Rad51 Recombinase/metabolismo , Recombinação Genética/genética , Espermatozoides/metabolismoRESUMO
The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Vetores Genéticos/genética , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Células NIH 3T3 , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11ß isoform-specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.
Assuntos
Pareamento Cromossômico , Troca Genética , Endodesoxirribonucleases/metabolismo , Meiose , Cromossomo X/fisiologia , Cromossomo Y/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Feminino , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Fosfato , Isoformas de Proteínas , Rad51 Recombinase/metabolismoRESUMO
Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11(-/ -)adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11(-/ -)and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.
Assuntos
Esterases/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Espermatócitos/citologia , Testículo/metabolismo , Animais , Endodesoxirribonucleases , Expressão Gênica , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Prófase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Maturidade Sexual , Espermatogênese/genéticaRESUMO
SPO11 introduces double-strand breaks (DSBs) that trigger the phosphorylation of H2AX during meiotic prophase. In mice, SPO11 is strictly required for initiation of meiotic recombination and synapsis, yet SPO11 is still considered to be dispensable for sex-body formation in mouse spermatocytes. We provide conclusive evidence showing that functional SPO11, and consequently recombination and synapsis, are required for phosphorylation of H2AX in the X-Y chromatin and for sex-body formation in mouse spermatocytes. We investigated the role in meiosis of the three kinases [ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia- and Rad-3-related) and DNA-PKcs (DNA-dependent-protein-kinase catalytic subunit)] known to phosphorylate H2AX in mitotic cells. We found that DNA-PKcs can be ruled out as an essential kinase in this process, whereas ATM is strictly required for the chromatin-wide phosphorylation of H2AX occurring in leptotene spermatocytes in response to DSBs. Remarkably, we discovered that Spo11 heterozygosity can rescue the prophase-I-arrest characteristic of ATM-deficient spermatocytes. Characterization of the rescued Atm-/- Spo11+/- mutant indicates that ATM is dispensable for sex-body formation and phosphorylation of H2AX in this subnuclear domain. The co-localization of ATR, phosphorylated H2AX and the sex chromatin observed in the Atm-/- Spo11+/- mutant, along with ATR transcription kinetics during the first wave of spermatogenesis, confirm and expand recent findings indicating that ATR is the kinase involved in H2AX phosphorylation in the sex body.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Heterozigoto , Prófase Meiótica I/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas/fisiologia , Cromatina Sexual/metabolismo , Espermatócitos/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico/genética , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Esterases , Expressão Gênica , Histonas/metabolismo , Masculino , Meiose/genética , Meiose/fisiologia , Prófase Meiótica I/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Espermatócitos/citologia , Testículo/citologia , Testículo/ultraestrutura , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/- mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.