RESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Regulación de la Expresión Génica , Histona Demetilasas/metabolismo , Histonas , Proteínas Represoras/metabolismo , Animales , Cromatina/genética , Proteínas Co-Represoras , Genoma , Células HEK293 , Células HeLa , Histona Demetilasas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteínas Represoras/genética , Células Sf9 , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Intercambio Genético , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Profase Meiótica I , Ratones/embriología , Ratones/metabolismo , Espermatogonias/metabolismo , Alelos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Masculino , Ratones/genética , ARN Helicasas , Recombinación Genética , Espermatogénesis , Espermatogonias/citología , Espermatogonias/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Evolución Molecular , Conversión Génica , Regiones Promotoras Genéticas , Recombinación Genética , Animales , Mapeo Cromosómico , Islas de CpG , Perros , Estudios de Asociación Genética , Genoma , N-Metiltransferasa de Histona-Lisina/genética , Desequilibrio de LigamientoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Cromosomas/genética , Exonucleasas , Meiosis/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Exonucleasas/genética , Exonucleasas/metabolismo , Células Germinativas/citología , Células Germinativas/metabolismo , Masculino , Ratones , Complejos Multiproteicos , Testículo/citología , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Inestabilidad Genómica , Recombinasas/genética , Espermatogénesis/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Masculino , Mamíferos/genética , Mamíferos/metabolismo , Profase Meiótica I , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasas/metabolismo , Recombinación Genética , Espermatocitos/metabolismo , Testículo/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81-/- animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Intercambio Genético , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Meiosis/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Homocigoto , Masculino , Ratones , Ratones Noqueados , Homólogo 1 de la Proteína MutL , Proteínas MutL , Mutación , Proteínas Nucleares/metabolismo , Oocitos/citología , Oocitos/metabolismo , Recuento de Espermatozoides , Testículo/citología , Testículo/metabolismoRESUMEN
During meiosis, induction of DNA double strand breaks (DSB) leads to recombination between homologous chromosomes, resulting in crossovers (CO) and non-crossovers (NCO). In the mouse, only 10% of DSBs resolve as COs, mostly through a class I pathway dependent on MutSγ (MSH4/ MSH5) and MutLγ (MLH1/MLH3), the latter representing the ultimate marker of these CO events. A second Class II CO pathway accounts for only a few COs, but is not thought to involve MutSγ/ MutLγ, and is instead dependent on MUS81-EME1. For class I events, loading of MutLγ is thought to be dependent on MutSγ, however MutSγ loads very early in prophase I at a frequency that far exceeds the final number of class I COs. Moreover, loss of MutSγ in mouse results in apoptosis before CO formation, preventing the analysis of its CO function. We generated a mutation in the ATP binding domain of Msh5 (Msh5GA ). While this mutation was not expected to affect MutSγ complex formation, MutSγ foci do not accumulate during prophase I. However, most spermatocytes from Msh5GA/GA mice progress to late pachynema and beyond, considerably further than meiosis in Msh5-/- animals. At pachynema, Msh5GA/GA spermatocytes show persistent DSBs, incomplete homolog pairing, and fail to accumulate MutLγ. Unexpectedly, Msh5GA/GA diakinesis-staged spermatocytes have no chiasmata at all from any CO pathway, indicating that a functional MutSγ complex is critical for all CO events regardless of their mechanism of generation.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Meiosis/genética , Complejos Multiproteicos/metabolismo , Mutación , Dominios Proteicos/genética , Adenosina Trifosfato/metabolismo , Animales , Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Masculino , Mamíferos , Ratones , Ratones Noqueados , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Espermatocitos/metabolismoRESUMEN
NIMA-related kinase 1 (NEK1) is a serine/threonine and tyrosine kinase that is highly expressed in mammalian germ cells. Mutations in Nek1 induce anemia, polycystic kidney and infertility. In this study we evaluated the role of NEK1 in meiotic spindle formation in both male and female gametes. Our results show that the lack of NEK1 provokes an abnormal organization of the meiosis I spindle characterized by elongated and/or multipolar spindles, and abnormal chromosome congression. The aberrant spindle structure is concomitant with the disruption in localization and protein levels of myosin X (MYO10) and α-adducin (ADD1), both of which are implicated in the regulation of spindle formation during mitosis. Interaction of ADD1 with MYO10 is dependent on phosphorylation, whereby phosphorylation of ADD1 enables its binding to MYO10 on mitotic spindles. Reduction in ADD1 protein in NEK1 mutant mice is associated with hyperphosphorylation of ADD1, thereby preventing the interaction with MYO10 during meiotic spindle formation. Our results reveal a novel regulatory role for NEK1 in the regulation of spindle architecture and function during meiosis.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Meiosis/fisiología , Miosinas/metabolismo , Quinasa 1 Relacionada con NIMA/fisiología , Huso Acromático/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Oocitos/ultraestructura , Fosforilación , Espermatocitos/ultraestructuraRESUMEN
Mammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, sororin, and PDS5B and direct cleavage by separase. Here, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by protein phosphatase 1 gamma (PP1γ), which both interacts with and is a phosphotarget of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ, leading to the dephosphorylation of WAPL, which, in turn, results in its retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/metabolismo , Meiosis , Quinasa 1 Relacionada con NIMA/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas/metabolismo , Animales , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Biológicos , Fenotipo , Fosforilación , Transducción de Señal , Espermatozoides/metabolismo , CohesinasRESUMEN
Meiotic crossovers (COs) are crucial for ensuring accurate homologous chromosome segregation during meiosis I. Because the double-strand breaks (DSBs) that initiate meiotic recombination greatly outnumber eventual COs, this process requires exquisite regulation to narrow down the pool of DSB intermediates that may form COs. In this paper, we identify a cyclin-related protein, CNTD1, as a critical mediator of this process. Disruption of Cntd1 results in failure to localize CO-specific factors MutLγ and HEI10 at designated CO sites and also leads to prolonged high levels of pre-CO intermediates marked by MutSγ and RNF212. These data show that maturation of COs is intimately coupled to deselection of excess pre-CO sites to yield a limited number of COs and that CNTD1 coordinates these processes by regulating the association between the RING finger proteins HEI10 and RNF212 and components of the CO machinery.
Asunto(s)
Intercambio Genético , Ciclinas/genética , Ciclinas/fisiología , Meiosis , Animales , Proteínas de Ciclo Celular , Diferenciación Celular , Segregación Cromosómica , Cromosomas/ultraestructura , Roturas del ADN de Doble Cadena , Ligasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación , Fenotipo , Recombinación Genética , Recuento de Espermatozoides , Espermatocitos/citología , Espermatocitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.
Asunto(s)
Intercambio Genético/genética , Ligasas/antagonistas & inhibidores , Meiosis/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular , Intercambio Genético/fisiología , Electroforesis en Gel de Poliacrilamida , Etiquetado Corte-Fin in Situ , Indoles , Ligasas/metabolismo , Masculino , Meiosis/fisiología , Ratones , Ratones Endogámicos C57BL , Proteína SUMO-1/metabolismo , Espermatocitos/citología , Espermatocitos/fisiología , Estadísticas no Paramétricas , Complejo Sinaptonémico/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Crossing-over ensures accurate chromosome segregation during meiosis, and every pair of chromosomes obtains at least one crossover, even though the majority of recombination sites yield non-crossovers. A putative regulator of crossing-over is RNF212, which is associated with variation in crossover rates in humans. We show that mouse RNF212 is essential for crossing-over, functioning to couple chromosome synapsis to the formation of crossover-specific recombination complexes. Selective localization of RNF212 to a subset of recombination sites is shown to be a key early step in the crossover designation process. RNF212 acts at these sites to stabilize meiosis-specific recombination factors, including the MutSγ complex (MSH4-MSH5). We infer that selective stabilization of key recombination proteins is a fundamental feature of meiotic crossover control. Haploinsufficiency indicates that RNF212 is a limiting factor for crossover control and raises the possibility that human alleles may alter the amount or stability of RNF212 and be risk factors for aneuploid conditions.
Asunto(s)
Intercambio Genético , Meiosis , Recombinación Genética , Ubiquitina-Proteína Ligasas/genética , Aneuploidia , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Compensación de Dosificación (Genética) , Humanos , Ligasas , RatonesRESUMEN
Bloom's syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS patients, is one of five mammalian RecQ helicases. Although BLM has been shown to promote genome stability by assisting in the repair of DNA structures that arise during homologous recombination in somatic cells, less is known about its role in meiotic recombination primarily because of the embryonic lethality associated with Blm deletion. However, the localization of BLM protein on meiotic chromosomes together with evidence from yeast and other organisms implicates a role for BLM helicase in meiotic recombination events, prompting us to explore the meiotic phenotype of mice bearing a conditional mutant allele of Blm. In this study, we show that BLM deficiency does not affect entry into prophase I but causes severe defects in meiotic progression. This is exemplified by improper pairing and synapsis of homologous chromosomes and altered processing of recombination intermediates, resulting in increased chiasmata. Our data provide the first analysis of BLM function in mammalian meiosis and strongly argue that BLM is involved in proper pairing, synapsis, and segregation of homologous chromosomes; however, it is dispensable for the accumulation of recombination intermediates.
Asunto(s)
Cromosomas de los Mamíferos/genética , Meiosis/genética , RecQ Helicasas/metabolismo , Recombinación Genética , Alelos , Animales , Proteínas Portadoras/metabolismo , Emparejamiento Cromosómico/genética , Femenino , Masculino , Profase Meiótica I/genética , Ratones , Ratones Noqueados , Ratones Mutantes , Proteínas MutL , Mutación , Fenotipo , RecQ Helicasas/deficiencia , RecQ Helicasas/genética , Espermatocitos/metabolismoRESUMEN
Meiotic recombination plays a key role in the maintenance of sequence diversity in the human genome. However, little is known about the fine-scale distribution and processes of recombination in human chromosomes, or how these impact on patterns of human diversity. We have therefore developed sperm typing systems that allow human recombination to be analysed at very high resolution. The emerging picture is that human crossovers are far from randomly distributed but instead are targeted into very narrow hot spots that can profoundly influence patterns of haplotype diversity in the human genome. These hot spots provide fundamental information on processes of human crossover and gene conversion, as well as evidence that they can violate basic rules of Mendelian inheritance.