RESUMO
In recent years, targeted genome editing has emerged as an indispensable tool for creating animal models, facilitating a comprehensive exploration of the molecular mechanisms governing a myriad of biological processes. Within this scientific landscape, the investigation of meiosis in mice has attracted considerable attention across numerous research laboratories. The precision and versatility of the CRISPR/Cas9 genome editing system have revolutionized our ability to generate mice with tailored genetic alterations, including point mutations and null mutations. These genetic modifications have provided invaluable insights into the intricate functionality of various meiotic genes and their associated variants. In this context, we present a detailed state of the art protocol for the creation of novel mouse models, each bearing specific genetic modifications within key meiotic genes, through the application of CRISPR/Cas9 technology. Furthermore, we showcase two distinct genetic modifications, accomplished within our laboratory, that can serve as valuable reference points for researchers seeking to elucidate the molecular intricacies of meiosis in mammals.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Meiose , Animais , Meiose/genética , Camundongos , Edição de Genes/métodos , Masculino , Modelos Animais , Feminino , RNA Guia de Sistemas CRISPR-Cas/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
The synaptonemal complex (SC) is a meiosis-specific multiprotein complex that forms between homologous chromosomes during prophase of meiosis I. Upon assembly, the SC mediates the synapses of the homologous chromosomes, leading to the formation of bivalents, and physically supports the formation of programmed double-strand breaks (DSBs) and their subsequent repair and maturation into crossovers (COs), which are essential for genome haploidization. Defects in the assembly of the SC or in the function of the associated meiotic recombination machinery can lead to meiotic arrest and human infertility. The majority of proteins and complexes involved in these processes are exclusively expressed during meiosis or harbor meiosis-specific subunits, although some have dual functions in somatic DNA repair and meiosis. Consistent with their functions, aberrant expression and malfunctioning of these genes have been associated with cancer development. In this review, we focus on the significance of the SC and their meiotic-associated proteins in human fertility, as well as how human genetic variants encoding for these proteins affect the meiotic process and contribute to infertility and cancer development.
Assuntos
Infertilidade , Meiose , Neoplasias , Complexo Sinaptonêmico , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Humanos , Meiose/genética , Neoplasias/genética , Infertilidade/genética , Masculino , Feminino , Recombinação GenéticaRESUMO
BACKGROUND: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. METHODS: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. RESULTS: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. CONCLUSIONS: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.
Assuntos
Doença da Válvula Aórtica Bicúspide , Cardiomiopatias , Cardiopatias Congênitas , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Cardiopatias Congênitas/complicações , Cardiomiopatias/etiologia , Miócitos Cardíacos , Valva Aórtica/diagnóstico por imagem , Fatores de Transcrição , Proteínas Cromossômicas não HistonaRESUMO
Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFß signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations. SIGNIFICANCE: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.
Assuntos
Tumor de Células da Granulosa , Neoplasias Ovarianas , Anormalidades da Pele , Adulto , Feminino , Humanos , Animais , Camundongos , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Ovarianas/genética , Mutação , Proteína Forkhead Box L2/genéticaRESUMO
Meiosis is a highly conserved specialized cell division process that generates haploid gametes. Many of its events are associated with dynamically regulated chromosomal structures and chromatin remodeling, which are mainly modulated by histone modifications. Histone H1 is a linker histone essential for packing the nucleosome into higher-order structures, and H1FOO (H1 histone family, member O, oocyte-specific) is a H1 variant whose expression pattern is restricted to growing oocytes and zygotes. To further explore the function of H1FOO, we generated mice lacking the H1foo gene by the CRISPR/Cas9 technique. Herein, we combine mouse genetics and cellular studies to show that H1foo-null mutants have no overt phenotype, with both males and females being fertile and presenting no gross defects in meiosis progression nor in synapsis dynamics. Accordingly, the histological sections show a normal development of gametes in both male and female mice. Considering the important role of oocyte constituents in enhancing mammalian somatic cell reprogramming, we analyzed iPSCs generation in H1foo mutant MEFs and observed no differences in the absence of H1FOO. Taken all together, in this work we present the first in vivo evidence of H1FOO dispensability for mouse fertility, clarifying the debate in the field surrounding its essentiality in meiosis.
Assuntos
Histonas , Oogênese , Feminino , Masculino , Camundongos , Animais , Oócitos , Fertilidade , Meiose , MamíferosRESUMO
Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.
Assuntos
Proteínas de Ligação a DNA , Insuficiência Ovariana Primária , Animais , Feminino , Humanos , Camundongos , Aberrações Cromossômicas , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Meiose , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/metabolismoRESUMO
BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.
Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Espermatogênese/fisiologia , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cristalografia por Raios X/métodos , Feminino , Recombinação Homóloga , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Meiose , Camundongos , Modelos Animais , Domínios e Motivos de Interação entre Proteínas , Deleção de SequênciaRESUMO
Histone deacetylase 11 (HDAC11) is the latest identified member of the histone deacetylase family of enzymes. It is highly expressed in brain, heart, testis, kidney, and skeletal muscle, although its role in these tissues is poorly understood. Here, we investigate for the first time the consequences of HDAC11 genetic impairment on skeletal muscle regeneration, a process principally dependent on its resident stem cells (satellite cells) in coordination with infiltrating immune cells and stromal cells. Our results show that HDAC11 is dispensable for adult muscle growth and establishment of the satellite cell population, while HDAC11 deficiency advances the regeneration process in response to muscle injury. This effect is not caused by differences in satellite cell activation or proliferation upon injury, but rather by an enhanced capacity of satellite cells to differentiate at early regeneration stages in the absence of HDAC11. Infiltrating HDAC11-deficient macrophages could also contribute to this accelerated muscle regenerative process by prematurely producing high levels of IL-10, a cytokine known to promote myoblast differentiation. Altogether, our results show that HDAC11 depletion advances skeletal muscle regeneration and this finding may have potential implications for designing new strategies for muscle pathologies coursing with chronic damage. DATABASE: Data were deposited in NCBI's Gene Expression Omnibus accessible through GEO Series accession number GSE147423.
Assuntos
Diferenciação Celular/genética , Histona Desacetilases/genética , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Proliferação de Células/genética , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Histona Desacetilases/metabolismo , Humanos , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , RNA-Seq/métodos , Regeneração/genética , Células Satélites de Músculo Esquelético/citologiaRESUMO
Skeletal muscle is the largest tissue in mammalian organisms and is a key determinant of basal metabolic rate and whole-body energy metabolism. Histone deacetylase 11 (HDAC11) is the only member of the class IV subfamily of HDACs, and it is highly expressed in skeletal muscle, but its role in skeletal muscle physiology has never been investigated. Here, we describe for the first time the consequences of HDAC11 genetic deficiency in skeletal muscle, which results in the improvement of muscle function enhancing fatigue resistance and muscle strength. Loss of HDAC11 had no obvious impact on skeletal muscle structure but increased the number of oxidative myofibers by promoting a glycolytic-to-oxidative muscle fiber switch. Unexpectedly, HDAC11 was localized in muscle mitochondria and its deficiency enhanced mitochondrial content. In particular, we showed that HDAC11 depletion increased mitochondrial fatty acid ß-oxidation through activating the AMP-activated protein kinase-acetyl-CoA carboxylase pathway and reducing acylcarnitine levels in vivo, thus providing a mechanistic explanation for the improved muscle strength and fatigue resistance. Overall, our data reveal a unique role of HDAC11 in the maintenance of muscle fiber-type balance and the mitochondrial lipid oxidation. These findings shed light on the mechanisms governing muscle metabolism and may have implications for chronic muscle metabolic disease management.
Assuntos
Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/genética , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Glicólise/genética , Histona Desacetilases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , OxirreduçãoRESUMO
Meiotic reductional division depends on the synaptonemal complex (SC), a supramolecular protein assembly that mediates homologous chromosomes synapsis and promotes crossover formation. The mammalian SC has eight structural components, including SYCE1, the only central element protein with known causative mutations in human infertility. We combine mouse genetics, cellular, and biochemical studies to reveal that SYCE1 undergoes multivalent interactions with SC component SIX6OS1. The N terminus of SIX6OS1 binds and disrupts SYCE1's core dimeric structure to form a 1:1 complex, while their downstream sequences provide a distinct second interface. These interfaces are separately disrupted by SYCE1 mutations associated with nonobstructive azoospermia and premature ovarian failure (POF), respectively. Mice harboring SYCE1's POF mutation and a targeted deletion within SIX6OS1's N terminus are infertile with failure of chromosome synapsis. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly, thus explaining how SYCE1's reported clinical mutations give rise to human infertility.
Assuntos
Azoospermia , Proteínas de Ligação a DNA , Animais , Azoospermia/genética , Pareamento Cromossômico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Mamíferos/genética , Camundongos , Mutação , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismoRESUMO
Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.
Assuntos
Proteínas de Transporte , Proteínas de Choque Térmico , Meiose/genética , Mutação de Sentido Incorreto/genética , Insuficiência Ovariana Primária/genética , Recombinação Genética/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Knockout , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Sequenciamento do ExomaRESUMO
Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin1-3. Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin1. In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions4,5. Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma6,7, is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2-MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin8,9, SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31comet liberate separase from SGO2-MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Securina , Separase/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Linhagem Celular , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Ligação Proteica , Securina/metabolismo , Separase/metabolismo , CoesinasRESUMO
The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.
Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Metáfase/genética , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Animais , Apoptose/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Complexo Sinaptonêmico/metabolismo , Testículo/citologia , Testículo/metabolismoRESUMO
Mammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Genômica/métodos , Espermatogênese/genética , Humanos , Masculino , Conformação Molecular , CoesinasRESUMO
Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.
Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , Prófase/fisiologia , Espermatócitos/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Centrômero/genética , Masculino , Camundongos , Camundongos Knockout , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espermatócitos/citologia , Fuso Acromático/genética , Fuso Acromático/metabolismo , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismoRESUMO
Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.
Assuntos
Cisteína Endopeptidases/genética , Fertilidade/genética , Gametogênese/genética , Fenótipo , Animais , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Marcação de Genes , Estudos de Associação Genética , Loci Gênicos , Células Germinativas/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Ovário/metabolismo , Testículo/metabolismoRESUMO
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RESUMO
Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.