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1.
Nucleic Acids Res ; 49(5): 2609-2628, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619545

RESUMO

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.


Assuntos
Quebras de DNA de Cadeia Dupla , Meiose/genética , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/fisiologia , Pareamento Cromossômico , Retroalimentação Fisiológica , Gametogênese , Camundongos , Estágio Paquíteno , Cromossomos Sexuais , Transdução de Sinais
2.
Nat Cell Biol ; 18(11): 1208-1220, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27723721

RESUMO

DNA double-strand breaks (DSBs) are induced by SPO11 during meiosis to initiate recombination-mediated pairing and synapsis of homologous chromosomes. Germline genome integrity requires spatiotemporal control of DSB formation, which involves the proteinaceous chromosome axis along the core of each meiotic chromosome. In particular, a component of unsynapsed axes, HORMAD1, promotes DSB formation in unsynapsed regions where DSB formation must occur to ensure completion of synapsis. Despite its importance, the underlying mechanism has remained elusive. We identify CCDC36 as a direct interactor of HORMAD1 (IHO1) that is essential for DSB formation. Underpinning this function, IHO1 and conserved SPO11-auxiliary proteins MEI4 and REC114 assemble chromatin-bound recombinosomes that are predicted activators of DSB formation. HORMAD1 is needed for robust recruitment of IHO1 to unsynapsed axes and efficient formation and/or stabilization of these recombinosomes. Thus, we propose that HORMAD1-IHO1 interaction provides a mechanism for the selective promotion of DSB formation along unsynapsed chromosome axes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromatina/genética , Replicação do DNA/genética , Endodesoxirribonucleases/metabolismo , Meiose , Camundongos Knockout , Proteínas Nucleares/metabolismo , Recombinação Genética/genética , Complexo Sinaptonêmico/genética
3.
Genes Dev ; 27(13): 1484-94, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23824539

RESUMO

In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Meiose , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Cromossomos/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Fosforilação , Transporte Proteico/genética , Proteínas Repressoras/metabolismo
4.
Genes Dev ; 26(9): 958-73, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22549958

RESUMO

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico/genética , Quebras de DNA de Cadeia Dupla , Animais , Proteínas de Ciclo Celular/genética , Feminino , Infertilidade Masculina/genética , Masculino , Meiose/genética , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Oócitos/metabolismo , Proteínas de Ligação a Fosfato , Complexo Sinaptonêmico/genética
5.
Mol Cell Biol ; 32(3): 633-51, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22144583

RESUMO

MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor ß (TGF-ß)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.


Assuntos
Neoplasias da Mama/patologia , Proteínas Fetais/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Neoplasias da Mama/metabolismo , Miosinas Cardíacas/biossíntese , Linhagem Celular Tumoral , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Feminino , Forminas , Regulação Neoplásica da Expressão Gênica , Humanos , Cadeias Leves de Miosina/biossíntese , Invasividade Neoplásica , Proteínas de Fusão Oncogênica/metabolismo , Fator de Resposta Sérica/biossíntese , Fibras de Estresse/metabolismo , Transativadores , Fator de Crescimento Transformador beta/metabolismo
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