RESUMO
Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.
Assuntos
Epigenômica , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Testículo/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Células Germinativas , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cromossomos Sexuais/genética , Espermatogênese , Testículo/citologia , Ubiquitina-Proteína Ligases/fisiologia , UbiquitinaçãoRESUMO
During meiosis, DNA damage response (DDR) proteins induce transcriptional silencing of unsynapsed chromatin, including the constitutively unsynapsed XY chromosomes in males. DDR proteins are also implicated in double strand break repair during meiotic recombination. Here, we address the function of the breast cancer susceptibility gene Brca1 in meiotic silencing and recombination in mice. Unlike in somatic cells, in which homologous recombination defects of Brca1 mutants are rescued by 53bp1 deletion, the absence of 53BP1 did not rescue the meiotic failure seen in Brca1 mutant males. Further, BRCA1 promotes amplification and spreading of DDR components, including ATR and TOPBP1, along XY chromosome axes and promotes establishment of pericentric heterochromatin on the X chromosome. We propose that BRCA1-dependent establishment of X-pericentric heterochromatin is critical for XY body morphogenesis and subsequent meiotic progression. In contrast, BRCA1 plays a relatively minor role in meiotic recombination, and female Brca1 mutants are fertile. We infer that the major meiotic role of BRCA1 is to promote the dramatic chromatin changes required for formation and function of the XY body.
Assuntos
Proteína BRCA1/fisiologia , Dano ao DNA , Heterocromatina/genética , Meiose , Recombinação Genética , Cromossomo X/genética , Alelos , Animais , Pareamento Cromossômico , Cromossomos/metabolismo , Éxons , Feminino , Deleção de Genes , Inativação Gênica , Masculino , Camundongos , Mutação , Fenótipo , EspermatogêneseRESUMO
The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HCT116 , Células HEK293 , Chaperonas de Histonas , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação/fisiologia , Ubiquitina/fisiologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/fisiologiaRESUMO
Sex chromosome inactivation is essential epigenetic programming in male germ cells. However, it remains largely unclear how epigenetic silencing of sex chromosomes impacts the evolution of the mammalian genome. Here we demonstrate that male sex chromosome inactivation is highly conserved between humans and mice and has an impact on the genetic evolution of human sex chromosomes. We show that, in humans, sex chromosome inactivation established during meiosis is maintained into spermatids with the silent compartment postmeiotic sex chromatin (PMSC). Human PMSC is illuminated with epigenetic modifications such as trimethylated lysine 9 of histone H3 and heterochromatin proteins CBX1 and CBX3, which implicate a conserved mechanism underlying the maintenance of sex chromosome inactivation in mammals. Furthermore, our analyses suggest that male sex chromosome inactivation has impacted multiple aspects of the evolutionary history of mammalian sex chromosomes: amplification of copy number, retrotranspositions, acquisition of de novo genes, and acquisition of different expression profiles. Most strikingly, profiles of escape genes from postmeiotic silencing diverge significantly between humans and mice. Escape genes exhibit higher rates of amino acid changes compared with non-escape genes, suggesting that they are beneficial for reproductive fitness and may allow mammals to cope with conserved postmeiotic silencing during the evolutionary past. Taken together, we propose that the epigenetic silencing mechanism impacts the genetic evolution of sex chromosomes and contributed to speciation and reproductive diversity in mammals.
Assuntos
Cromatina/genética , Cromossomos Humanos Y/genética , Evolução Molecular , Meiose , Animais , Cromatina/metabolismo , Homólogo 5 da Proteína Cromobox , Cromossomos Humanos Y/metabolismo , Epigênese Genética , Dosagem de Genes , Perfilação da Expressão Gênica , Genes Ligados ao Cromossomo X , Humanos , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Espermatogênese/genética , Espermatogônias/metabolismo , Transcrição GênicaRESUMO
Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.
Assuntos
Dano ao DNA/genética , Inativação do Cromossomo X/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Dano ao DNA/fisiologia , Reparo do DNA , Epigênese Genética , Feminino , Células Germinativas/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Meiose/genética , Meiose/fisiologia , Modelos Biológicos , Modelos Genéticos , Transdução de Sinais , Inativação do Cromossomo X/fisiologiaRESUMO
Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.
Assuntos
Regulação para Baixo , Fibroblastos/metabolismo , Histonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Western Blotting , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Citotoxinas/farmacologia , Diploide , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Histonas/genética , Camundongos , Camundongos Knockout , Poliploidia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Zinostatina/farmacologiaRESUMO
Chromosome-wide inactivation is an epigenetic signature of sex chromosomes. The mechanism by which the chromosome-wide domain is recognized and gene silencing is induced remains unclear. Here we identify an essential mechanism underlying the recognition of the chromosome-wide domain in the male germline. We show that mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX), defines the chromosome-wide domain, initiates meiotic sex chromosome inactivation (MSCI), and leads to XY body formation. Importantly, MSCI consists of two genetically separable steps. The first step is the MDC1-independent recognition of the unsynapsed axis by DNA damage response (DDR) factors such as ataxia telangiectasia and Rad3-related (ATR), TOPBP1, and γH2AX. The second step is the MDC1-dependent chromosome-wide spreading of DDR factors to the entire chromatin. Furthermore, we demonstrate that, in somatic cells, MDC1-dependent amplification of the γH2AX signal occurs following replicative stress and is associated with transcriptional silencing. We propose that a common DDR pathway underlies both MSCI and the response of somatic cells to replicative stress. These results establish that the DDR pathway centered on MDC1 triggers epigenetic silencing of sex chromosomes in germ cells.
Assuntos
Mecanismo Genético de Compensação de Dose/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cromossomos Sexuais/genética , Espermatozoides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Feminino , Heterocromatina/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recombinação Genética , Testículo/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.
Assuntos
Dano ao DNA , Replicação do DNA , Mitose , Poliploidia , Animais , Divisão Celular , Transformação Celular Neoplásica , Células Cultivadas , Camundongos , Oncogenes , Fase SRESUMO
Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication-mediated DNA double-strand breaks (DSBs). Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA-PK-dependent manner. To distinguish the roles of phosphatidylinositol 3-kinase-related protein kinases including DNA-PK, ataxia-telangiectasia mutated (ATM), and ATR, in the response to replication-mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock-down with siRNA suppressed CPT-induced RPA2 hyperphosphorylation and focus formation. CPT-induced RPA2 focus formation was normally observed in DNA-PK- or ATM-deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA-PK-dependent in CPT-treated cells and DNA-PK-independent in HU-treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU-treated cells occurred later than in the CPT-treated cells, indicating that the DNA-PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA-PK is responsible for the RPA2 hyperphosphorylation following ATR-dependent RPA2 focus formation in response to replication-mediated DSBs directly induced by CPT.
Assuntos
Dano ao DNA , Replicação do DNA , DNA/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Camptotecina/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxiureia/farmacologia , Rim/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Proteína de Replicação A/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (gamma-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-gamma-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia-telangiectasia cells lacking ATM kinase activity, gamma-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity.
Assuntos
Dano ao DNA , Histonas/metabolismo , Mitose , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cromossomos Humanos/enzimologia , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA ligase IV is thought to be involved in DNA double-strand break repair and DNA non-homologous end-joining pathways, but these mechanisms are still unclear. To investigate the roles of DNA ligase IV from a biologically functional viewpoint, the authors studied its relationship to meiosis in a basidiomycete, Coprinus cinereus, which shows a highly synchronous meiotic cell cycle. The C. cinereus cDNA homologue of DNA ligase IV (CcLIG4) was successfully cloned. The 3.2 kb clone including the ORF encoded a predicted product of 1025 amino acid residues with a molecular mass of 117 kDa. A specific inserted sequence composed of 95 amino acids rich in aspartic acid and glutamic acid could be detected between tandem BRCT domains. The inserted sequence had no sequence identity with other eukaryotic counterparts of DNA ligase IV or with another aspartic acid and glutamic acid rich sequence inserted in C. cinereus proliferating cell nuclear antigen (CcPCNA), although the length and the percentages of aspartic and glutamic acids were similar. In addition, the recombinant CcLIG4 protein not only showed ATP-dependent ligase activity, but also used (dT)(16)/poly(dA) and (dT)(16)/poly(rA) as substrates, and had double-strand ligation activity, like human DNA ligase IV. Northern hybridization analysis and in situ hybridization indicated that CcLIG4 was expressed not only at the pre-meiotic S phase but also at meiotic prophase I. Intense signals were observed in leptotene and zygotene. Based on these observations, the possible role(s) of C. cinereus DNA ligase IV during meiosis are discussed.
Assuntos
Coprinus/enzimologia , DNA Ligases/metabolismo , Alquilantes/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Coprinus/citologia , Coprinus/genética , DNA Ligase Dependente de ATP , DNA Ligases/química , DNA Ligases/genética , Reparo do DNA , DNA Complementar/genética , DNA Fúngico/genética , Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Humanos , Meiose , Metanossulfonato de Metila/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.