RESUMO
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
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
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
An Amendment to this Article has been published and is linked from the HTML version of this paper.
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.
Assuntos
Transformação Celular Neoplásica/metabolismo , Quebras de DNA de Cadeia Dupla , Interfase , Proteínas de Neoplasias/metabolismo , Reparo de DNA por Recombinação , Separase/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ativação Enzimática , Células HEK293 , Humanos , Ligases/genética , Ligases/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Separase/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controleRESUMO
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
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
The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Cinetocoros/metabolismo , Meiose , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Feminino , Humanos , Infertilidade/genética , Infertilidade/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Quinase 1 Polo-LikeRESUMO
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
The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1ß. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Complexo Sinaptonêmico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Espermatócitos/metabolismo , CoesinasRESUMO
Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Mutação , Proteínas Nucleares/genética , Insuficiência Ovariana Primária/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Infertilidade Feminina/genética , Camundongos , Linhagem , CoesinasRESUMO
Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3 encodes a meiosis-specific subunit of the cohesin complex, a large proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss-of-function mouse model for STAG3 in oogenesis. However, and since none of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement in male infertility. In this report, we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair, demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility.
Assuntos
Infertilidade Masculina/genética , Proteínas Nucleares/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Masculino , Meiose/genética , Meiose/fisiologia , Camundongos , Proteínas Nucleares/genética , Proteínas de Ligação a Fosfato , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Complexo Sinaptonêmico/metabolismo , CoesinasRESUMO
Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mamíferos/metabolismo , Meiose , Anáfase/efeitos dos fármacos , Animais , Centrômero/efeitos dos fármacos , Cromátides/efeitos dos fármacos , Cromátides/metabolismo , Etoposídeo/farmacologia , Masculino , Meiose/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitose/efeitos dos fármacos , CoesinasRESUMO
Sister chromatids are held together by a protein complex named cohesin. Shugoshin proteins protect cohesin from cleavage by separase during meiosis I in eukaryotes and from phosphorylation-mediated removal during mitosis in vertebrates. This protection is crucial for chromosome segregation during mitosis and meiosis. Mechanistically, shugoshins shield cohesin by forming a complex with the phosphatase PP2A, which dephosphorylates cohesin, leading to its retention at centromeres during the onset of meiotic anaphase and vertebrate mitotic prophase I. In addition to this canonical function, shugoshins have evolved novel, species-specific cellular functions, the mechanisms of which remain a subject of intense debate, but are likely to involve spatio-temporally coordinated interactions with the chromosome passenger complex, the spindle checkpoint and the anaphase promoting complex. Here, we compare and contrast these remarkable features of shugoshins in model organisms and humans.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Anáfase , Animais , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Humanos , Mitose , Fosforilação , CoesinasRESUMO
The cohesin complex is a ring-shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α-kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α-kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I, which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L-deficient females are fertile but develop an age-dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Fatores Etários , Animais , Proteínas Cromossômicas não Histona/deficiência , Cromossomos/metabolismo , Feminino , Histocitoquímica , Infertilidade , Masculino , Camundongos , Camundongos Knockout , Ovário/patologia , Subunidades Proteicas/metabolismo , Fatores Sexuais , Testículo/patologiaRESUMO
Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos/genética , Meiose/fisiologia , Mitose/fisiologia , Células 3T3 , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Células Germinativas , Humanos , Mamíferos , CamundongosRESUMO
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
BACKGROUND: Ischemia in the placenta is considered the base of the pathogenesis of preeclampsia, a pregnancy-specific syndrome in which soluble endoglin (sEng) is a prognostic marker and plays a pathogenic role. Here, we investigated the effects of hypoxia and the downstream pathways in the release of sEng. METHODS AND RESULTS: Under hypoxic conditions, the trophoblast-like cell line JAR showed an increase in sEng parallel to an elevated formation of reactive oxygen species. Because reactive oxygen species are related to the formation of oxysterols, we assessed the effect of 22-(R)-hydroxycholesterol, a natural ligand of the liver X receptor (LXR), and the LXR synthetic agonist T0901317. Treatment of JAR cells or human placental explants with 22-(R)-hydroxycholesterol or T0901317 resulted in a clear increase in sEng that was dependent on LXR. These LXR agonists induced an increased matrix metalloproteinase-14 expression and activity and a significant reduction of its endogenous inhibitor, tissue inhibitor of metalloproteinase-3. In addition, mice treated with LXR agonists underwent an increase in the plasma sEng levels, concomitant with an increase in arterial pressure. Moreover, transgenic mice overexpressing sEng displayed high blood pressure. Finally, administration of an endoglin peptide containing the consensus matrix metalloproteinase-14 cleavage site G-L prevented the oxysterol-dependent increase in arterial pressure and sEng levels in mice. CONCLUSIONS: These studies provide a clue to the involvement of the LXR pathway in sEng release and its pathogenic role in vascular disorders such as preeclampsia.
Assuntos
Antígenos CD/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isquemia/metabolismo , Doenças Placentárias/metabolismo , Pré-Eclâmpsia/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Pressão Sanguínea/fisiologia , Linhagem Celular Tumoral , Coriocarcinoma , Endoglina , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrocarbonetos Fluorados/farmacologia , Hidroxicolesteróis/farmacologia , Isquemia/patologia , Receptores X do Fígado , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Nucleares Órfãos/metabolismo , Doenças Placentárias/patologia , Pré-Eclâmpsia/patologia , Gravidez , Sulfonamidas/farmacologia , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Neoplasias UterinasRESUMO
Matrix metalloproteinases (MMPs) have fundamental roles in tumor progression, but most clinical trials with MMP inhibitors have not shown improvements in individuals with cancer. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8(-/-)mice. Female Mmp8(-/-)mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.