RESUMO
Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.
Assuntos
Meiose , Prófase Meiótica I , Animais , Masculino , Camundongos , Alelos , DNA Helicases/genética , Reparo do DNA/genética , Meiose/genética , Prófase Meiótica I/genéticaRESUMO
Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.
Assuntos
Pareamento Cromossômico , Troca Genética , Meiose , Ubiquitina-Proteína Ligases , Animais , Camundongos , Masculino , Feminino , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Camundongos Knockout , Humanos , LigasesRESUMO
Meiosis, the mechanism of creating haploid gametes, is a complex cellular process observed across sexually reproducing organisms. Fundamental to meiosis is the process of homologous recombination, whereby DNA double-strand breaks are introduced into the genome and are subsequently repaired to generate either noncrossovers or crossovers. Although homologous recombination is essential for chromosome pairing during prophase I, the resulting crossovers are critical for maintaining homolog interactions and enabling accurate segregation at the first meiotic division. Thus, the placement, timing, and frequency of crossover formation must be exquisitely controlled. In this review, we discuss the proteins involved in crossover formation, the process of their formation and designation, and the rules governing crossovers, all within the context of the important landmarks of prophase I. We draw together crossover designation data across organisms, analyze their evolutionary divergence, and propose a universal model for crossover regulation.
Assuntos
Troca Genética , Quebras de DNA de Cadeia Dupla , Meiose , Aneuploidia , Animais , Reparo do DNA , Prófase Meiótica I , Processamento de Proteína Pós-Traducional , Recombinação Genética , Complexo Sinaptonêmico/fisiologiaRESUMO
DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.
Assuntos
Enzimas Reparadoras do DNA , Reparo do DNA , Exodesoxirribonucleases , Neoplasias , Animais , Linfócitos B , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Imunidade , Meiose/genética , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Hipermutação Somática de ImunoglobulinaRESUMO
Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.
Assuntos
Reparo do DNA , Endonucleases , Doença de Huntington/genética , Proteínas MutL , Processamento de Proteína , Expansão das Repetições de Trinucleotídeos , Animais , Células Cultivadas , Endonucleases/fisiologia , Feminino , Fibroblastos , Técnicas de Introdução de Genes , Instabilidade Genômica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas MutL/fisiologia , OligonucleotídeosRESUMO
Spermatogenesis is the process by which male gametes are formed from a self-renewing population of spermatogonial stem cells (SSCs) residing in the testis. SSCs represent less than 1% of the total testicular cell population in adults, but must achieve a stable balance between self-renewal and differentiation. Once differentiation has occurred, the newly formed and highly proliferative spermatogonia must then enter the meiotic program in which DNA content is doubled, then halved twice to create haploid gametes. While much is known about the critical cellular processes that take place during the specialized cell division that is meiosis, much less is known about how the spermatocytes in the "first-wave" in juveniles compare to those that contribute to long-term, "steady-state" spermatogenesis in adults. Given the strictly-defined developmental process of spermatogenesis, this study explored the transcriptional profiles of developmental cell stages during testis maturation. Using a combination of comprehensive germ cell sampling with high-resolution, single-cell-mRNA-sequencing, we have generated a reference dataset of germ cell gene expression. We show that discrete developmental stages of spermatogenesis possess significant differences in the transcriptional profiles from neonates compared to juveniles and adults. Importantly, these gene expression dynamics are also reflected at the protein level in their respective cell types. We also show differential utilization of many biological pathways with age in both spermatogonia and spermatocytes, demonstrating significantly different underlying gene regulatory programs in these cell types over the course of testis development and spermatogenic waves. This dataset represents the first unbiased sampling of spermatogonia and spermatocytes during testis maturation, at high-resolution, single-cell depth. Not only does this analysis reveal previously unknown transcriptional dynamics of a highly transitional cell population, it has also begun to reveal critical differences in biological pathway utilization in developing spermatogonia and spermatocytes, including response to DNA damage and double-strand breaks.
Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Análise de Célula Única/métodos , Espermatogênese/genética , Animais , Animais Recém-Nascidos/genética , Diferenciação Celular , Perfilação da Expressão Gênica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Diferenciação Sexual , Espermatócitos/fisiologia , Espermatogônias/fisiologia , Testículo/embriologia , Testículo/fisiologia , Transcriptoma/genéticaRESUMO
During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.
Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Prófase Meiótica I/genética , Proteínas MutL/genética , Animais , Pareamento Cromossômico/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Homóloga/genética , Masculino , Meiose/genética , Camundongos , Proteína 1 Homóloga a MutL/genética , Proteínas MutS/genética , Rad51 Recombinase/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismoRESUMO
SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1-LSD1-CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Histonas , Proteínas Repressoras/metabolismo , Animais , Cromatina/genética , Proteínas Correpressoras , Genoma , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Proteínas Repressoras/genética , Células Sf9 , Espermatogênese/genética , Testículo/metabolismoRESUMO
MicroRNAs and siRNAs, both of which are AGO-bound small RNAs, are essential for mammalian spermatogenesis. Although their precise germline roles remain largely uncharacterized, recent discoveries suggest that they function in mechanisms beyond microRNA-mediated post-transcriptional control, playing roles in DNA repair and transcriptional regulation within the nucleus. Here, we discuss the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline. We integrate genetic, clinical and genomics data, and draw upon findings from non-mammalian models, to examine potential roles for AGO-bound small RNAs during spermatogenesis. Finally, we evaluate the emerging and differing roles for AGOs and AGO-bound small RNAs in the male and female germlines, suggesting potential reasons for these sexual dimorphisms.
Assuntos
MicroRNAs/metabolismo , Espermatogênese/fisiologia , Animais , Feminino , Células Germinativas/metabolismo , Humanos , Masculino , MicroRNAs/genética , RNA Interferente Pequeno/genética , Espermatogênese/genéticaRESUMO
Telomeres are dynamic nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. They consist of telomeric DNA repeats (TTAGGG), the shelterin protein complex, and telomeric repeat-containing RNA (TERRA). Proposed TERRA functions are wide ranging and include telomere maintenance, telomerase inhibition, genomic stability, and alternative lengthening of telomere. However, the presence and role of TERRA in primordial germ cells (PGCs), the embryonic precursors of germ cells, is unknown. Using RNA-fluorescence in situ hybridization, we identify TERRA transcripts in female PGCs at 11.5, 12.5, and 13.5 days postcoitum. In male PGCs, the earliest detection TERRA was at 12.5 dpc where we observed cells with either zero or one TERRA focus. Using qRT-PCR, we evaluated chromosome-specific TERRA expression. Female PGCs showed TERRA expression at 11.5 dpc from eight different chromosome subtelomeric regions (chromosomes 1, 2, 7, 9, 11, 13, 17, and 18) while in male PGCs, TERRA expression was confined to the chromosome 17. Most TERRA transcription in 13.5 dpc male PGCs arose from chromosomes 2 and 6. TERRA interacting proteins were evaluated using identification of direct RNA interacting proteins (iDRiP), which identified 48 in female and 26 in male protein interactors from PGCs at 13.5 dpc. We validated two different proteins: the splicing factor, proline- and glutamine-rich (SFPQ) in PGCs and non-POU domain-containing octamer-binding protein (NONO) in somatic cells. Taken together, our data indicate that TERRA expression and interactome during PGC development are regulated in a dynamic fashion that is dependent on gestational age and sex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Germinativas Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Ligação Proteica , Fatores Sexuais , Distribuição Tecidual , Fatores de Transcrição/genéticaRESUMO
Fancj, the gene associated with Fanconi anemia (FA) Complementation Group J, encodes a DNA helicase involved in homologous recombination repair and the cellular response to replication stress. FANCJ functions in part through its interaction with key DNA repair proteins, including MutL homolog-1 (MLH1), Breast Cancer Associated gene-1 (BRCA1), and Bloom syndrome helicase (BLM). All three of these proteins are involved in a variety of events that ensure genome stability, including the events of DNA double strand break (DSB) repair during prophase I of meiosis. Meiotic DSBs are repaired through homologous recombination resulting in non-crossovers (NCO) or crossovers (CO). The frequency and placement of COs are stringently regulated to ensure that each chromosome receives at least one CO event, and that longer chromosomes receive at least one additional CO, thus facilitating the accurate segregation of homologous chromosomes at the first meiotic division. In the present study, we investigated the role of Fancj during prophase I using a gene trap mutant allele. Fancj (GT/GT) mutants are fertile, but their testes are very much smaller than wild-type littermates, predominantly as a result of impeded spermatogonial proliferation and mildly increased apoptosis during testis development in the fetus. This defect in spermatogonial proliferation is consistent with mutations in other FA genes. During prophase I, early events of synapsis and DSB induction/repair appear mostly normal in Fancj (GT/GT) males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in Fancj (GT/GT) males, indicative of increased DSB repair via CO, and is concomitant with increased chiasmata at diakinesis. This increase in COs in the absence of FANCJ is associated with increased localization of BLM helicase protein, indicating that BLM may facilitate the increased rate of crossing over in Fancj (GT/GT) males. Taken together, these results demonstrate a critical role for FANCJ in spermatogenesis at two stages: firstly in the proliferative activity that gives rise to the full complement of testicular spermatogonia and secondly in the establishment of appropriate CO numbers during prophase I.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Troca Genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Prófase Meiótica I , Camundongos/embriologia , Camundongos/metabolismo , Espermatogônias/metabolismo , Alelos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Masculino , Camundongos/genética , RNA Helicases , Recombinação Genética , Espermatogênese , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimentoRESUMO
Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3'UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1-ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , RNA Helicases DEAD-box/fisiologia , Meiose/fisiologia , MicroRNAs/genética , Proteínas de Ligação a RNA/fisiologia , Ribonuclease III/fisiologia , Cromossomos Sexuais/fisiologia , Espermatócitos/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatócitos/citologiaRESUMO
MicroRNAs are essential for spermatogenesis. However, the stage-specific requirements for particular miRNAs in the male mammalian germ line remain largely uncharacterized. The miR-34 family is, to date, the only miRNA proven to be necessary for the production of sperm in mammals, though its germline roles are poorly understood. Here, we generate and analyze paired small RNA and mRNA profiles across different stages of germline development in male mice, focusing on time points shortly before and during meiotic prophase I. We show that in addition to miR-34, miR-29 also mediates widespread repression of mRNA targets during meiotic prophase I in the male mouse germline. Furthermore, we demonstrate that predicted miR-29 target mRNAs in meiotic cells are largely distinct from those of miR-34, indicating that miR-29 performs a regulatory function independent of miR-34. Prior to this work, no germline role has been attributed to miR-29. To begin to understand roles for miR-29 in the germ line, we identify targets of miR-29 undergoing post transcriptional downregulation during meiotic prophase I, which likely correspond to the direct targets of miR-29. Interestingly, candidate direct targets of miR-29 are enriched in transcripts encoding extracellular matrix components. Our results implicate the miR-29 family as an important regulatory factor during male meiosis.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Meiose/genética , MicroRNAs/genética , Transcriptoma , Animais , Cílios/genética , Análise por Conglomerados , Matriz Extracelular/metabolismo , Redes Reguladoras de Genes , Masculino , Camundongos , Interferência de RNA , Transdução de Sinais , Espermatogênese/genéticaRESUMO
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.
Assuntos
Proteínas de Ciclo Celular , Cromossomos/genética , Exonucleases , Meiose/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Exonucleases/genética , Exonucleases/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Camundongos , Complexos Multiproteicos , Testículo/citologia , Testículo/metabolismoRESUMO
The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSß (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Doença de Huntington/genética , Proteínas Nucleares/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Modelos Animais de Doenças , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Instabilidade Genômica , Humanos , Doença de Huntington/patologia , Camundongos , Proteína 1 Homóloga a MutL , Proteínas MutL , RNA MensageiroRESUMO
The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.
Assuntos
Evolução Molecular , Conversão Gênica , Regiões Promotoras Genéticas , Recombinação Genética , Animais , Mapeamento Cromossômico , Ilhas de CpG , Cães , Estudos de Associação Genética , Genoma , Histona-Lisina N-Metiltransferase/genética , Desequilíbrio de LigaçãoRESUMO
The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm(-/-) males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm(-/-) males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM.
Assuntos
Instabilidade Genômica , Recombinases/genética , Espermatogênese/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Masculino , Mamíferos/genética , Mamíferos/metabolismo , Prófase Meiótica I , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recombinases/metabolismo , Recombinação Genética , Espermatócitos/metabolismo , Testículo/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis, and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted, including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.
Assuntos
Infertilidade Masculina , Meiose , Animais , Humanos , Masculino , Camundongos , Alelos , Proteínas de Transporte/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Infertilidade Masculina/genética , Proteínas Nucleares/genética , Cromossomos SexuaisRESUMO
MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis. The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2 6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process. Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis. Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice. To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3(-/-) mice. Here we show that Mlh3(-/-) mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I. Mlh3(-/-) spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization. Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.
Assuntos
Aneuploidia , Proteínas de Transporte/genética , Enzimas Reparadoras do DNA , Infertilidade Feminina/genética , Meiose , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Apoptose/genética , Proteínas de Transporte/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons , Feminino , Fertilização in vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Repetições de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteínas MutL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Oócitos/patologia , Análise de Sequência , Espermatócitos/metabolismo , Testículo/anormalidadesRESUMO
Meiosis is characterized by highly regulated transitions in gene expression that require diverse mechanisms of gene regulation. For example, in male mammals, transcription undergoes a global shut-down in early prophase I of meiosis, followed by increasing transcriptional activity into pachynema. Later, as spermiogenesis proceeds, the histones bound to DNA are replaced with transition proteins, which are themselves replaced with protamines, resulting in a highly condensed nucleus with repressed transcriptional activity. In addition, two specialized gene silencing events take place during prophase I: meiotic silencing of unsynapsed chromatin (MSUC), and the sex chromatin specific mechanism, meiotic sex chromosome inactivation (MSCI). Notably, conserved roles for the RNA binding protein (RBP) machinery that functions with small non-coding RNAs have been described as participating in these meiosis-specific mechanisms, suggesting that RNA-mediated gene regulation is critical for fertility in many species. Here, we review roles of small RNAs and their associated RBPs in meiosis-related processes such as centromere function, silencing of unpaired chromatin and meiotic recombination. We will discuss the emerging evidence of non-canonical functions of these components in meiosis.