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1.
Mol Biol Cell ; 35(8): ar105, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38865189

RESUMO

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and coorientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and biorientation of homologues, which are critical for accurate chromosome segregation in meiosis I.


Assuntos
Segregação de Cromossomos , Proteínas de Drosophila , Cinetocoros , Meiose , Microtúbulos , Oócitos , Cinetocoros/metabolismo , Animais , Meiose/fisiologia , Oócitos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Feminino , Centrômero/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Aurora Quinase B/metabolismo , Aurora Quinase B/genética
2.
bioRxiv ; 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38559067

RESUMO

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that SPC105R's C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for two activities that are critical for accurate chromosome segregation in meiosis I, lateral microtubule attachments and bi-orientation of homologs.

3.
Curr Biol ; 33(17): 3759-3765.e3, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37582374

RESUMO

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A.1 In cultured somatic cells, an established paradigm of cell-cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germ line challenges this model because of the cell-cycle arrest between pre-meiotic S phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocytes,2,3 suggesting that a similar process may be required for centromere inheritance in mammals. To test this hypothesis, we developed an oocyte-specific conditional knockout (cKO) mouse for Mis18α, an essential component of the assembly machinery. We find that embryos derived from Mis18α knockout oocytes fail to assemble CENP-A nucleosomes prior to zygotic genome activation (ZGA), validating the knockout model. We show that deletion of Mis18α in the female germ line at the time of birth has no impact on centromeric CENP-A nucleosome abundance, even after 6-8 months of aging. In addition, there is no detectable detriment to fertility. Thus, centromere chromatin is maintained long-term, independent of new assembly during the extended prophase I arrest in mouse oocytes.


Assuntos
Proteínas Cromossômicas não Histona , Nucleossomos , Feminino , Animais , Camundongos , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/genética , Centrômero/metabolismo , Cromatina , Oócitos/metabolismo , Envelhecimento , Autoantígenos , Mamíferos/genética
4.
bioRxiv ; 2023 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-37292821

RESUMO

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A 1 . In cultured somatic cells, an established paradigm of cell cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germline challenges this model because of the cell cycle arrest between pre-meiotic S-phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocyte 2,3 , suggesting that a similar process may be required for centromere inheritance in mammals. However, we show that centromere chromatin is maintained long-term independent of new assembly during the extended prophase I arrest in mouse oocytes. Conditional knockout of Mis18α, an essential component of the assembly machinery, in the female germline at the time of birth has almost no impact on centromeric CENP-A nucleosome abundance nor any detectable detriment to fertility.

5.
Hum Reprod ; 38(2): 195-203, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36478237

RESUMO

The post-reproductive phase or menopause in females is triggered by a physiological timer that depends on a threshold of follicle number in the ovary. Curiously, reproductive senescence appears to be decoupled from chronological age and is instead thought to be a function of physiological ageing. Ovarian ageing is associated with a decrease in oocyte developmental competence, attributed to a concomitant increase in meiotic errors. Although many biological hallmarks of general ageing are well characterized, the precise mechanisms underlying the programmed ageing of the female reproductive system remain elusive. In particular, the molecular pathways linking the external menopause trigger to the internal oocyte chromosome segregation machinery that controls fertility outcomes is unclear. However, recent large scale genomics studies have begun to provide insights into this process. Next-generation sequencing integrated with systems biology offers the advantage of sampling large datasets to uncover molecular pathways associated with a phenotype such as ageing. In this mini-review, we discuss findings from these studies that are crucial for advancing female reproductive senescence research. Targets identified in these studies can inform future animal models for menopause. We present three potential hypotheses for how external pathways governing ovarian ageing can influence meiotic chromosome segregation, with evidence from both animal models and molecular targets revealed from genomics studies. Although still in incipient stages, we discuss the potential of genomics studies combined with epigenetic age acceleration models for providing a predictive toolkit of biomarkers controlling menopause onset in women. We also speculate on future research directions to investigate extending female reproductive lifespan, such as comparative genomics in model systems that lack menopause. Novel genomics insights from such organisms are predicted to provide clues to preserving female fertility.


Assuntos
Envelhecimento , Reprodução , Animais , Feminino , Reprodução/genética , Envelhecimento/genética , Menopausa/genética , Fertilidade/genética , Genômica
6.
Nat Cell Biol ; 24(5): 748-756, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35534577

RESUMO

Centromeres are defined epigenetically by the histone H3 variant CENP-A. The propagation cycle by which pre-existing CENP-A nucleosomes serve as templates for nascent assembly predicts the epigenetic memory of weakened centromeres. Using a mouse model with reduced levels of CENP-A nucleosomes, we find that an embryonic plastic phase precedes epigenetic memory through development. During this phase, nascent CENP-A nucleosome assembly depends on the maternal Cenpa genotype rather than the pre-existing template. Weakened centromeres are thus limited to a single generation, and parental epigenetic differences are eliminated by equal assembly on maternal and paternal centromeres. These differences persist, however, when the underlying DNA of parental centromeres differs in repeat abundance, as assembly during the plastic phase also depends on sufficient repetitive centromere DNA. With contributions of centromere DNA and the Cenpa maternal effect, we propose that centromere inheritance naturally minimizes fitness costs associated with weakened centromeres or epigenetic differences between parents.


Assuntos
Herança Materna , Nucleossomos , Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Histonas/genética , Histonas/metabolismo , Herança Materna/genética , Nucleossomos/genética , Plásticos
7.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34297127

RESUMO

Meiosis in female oocytes lacks centrosomes, the microtubule-organizing centers. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, we examined the role of protein phosphatase 2A (PP2A) in Drosophila oocyte meiosis. We found that both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which has a B subunit encoded by two partially redundant paralogs, wdb and wrd, is also required for maintenance of sister chromatid cohesion, establishment of end-on microtubule attachments, and metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332 and kinetochore protein SPC105R. Although BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. We propose at least three populations of PP2A-B56 regulate meiosis, two of which depend on SPC105R and a third that is associated with the spindle.


Assuntos
Proteínas de Drosophila , Cinetocoros , Proteína Fosfatase 2 , Fuso Acromático , Animais , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Drosophila , Proteínas de Drosophila/genética , Feminino , Meiose , Microtúbulos , Oócitos , Proteína Fosfatase 2/genética
8.
Curr Top Dev Biol ; 140: 35-54, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32591081

RESUMO

The centromere directs chromosome segregation but is not itself genetically encoded. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant CENP-A, independent of the underlying DNA sequence. Therefore, to maintain centromeres and ensure accurate chromosome segregation, CENP-A nucleosomes must be inherited across generations through the germline. In this chapter we discuss three aspects of maternal centromere inheritance. First, we propose mechanisms for maintaining CENP-A nucleosomes through the prolonged prophase arrest in mammalian oocytes. Second, we review mechanisms by which selfish centromeres bias their transmission through female meiosis. Third, we discuss regulation of centromere size through early embryonic development.


Assuntos
Centrômero/genética , Desenvolvimento Embrionário/genética , Herança Materna/genética , Meiose/genética , Oócitos/metabolismo , Animais , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Epigênese Genética , Feminino , Nucleossomos/genética , Nucleossomos/metabolismo , Oócitos/citologia
9.
PLoS Genet ; 15(5): e1008072, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31150390

RESUMO

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.


Assuntos
Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiose , Microtúbulos/metabolismo , Proteína Fosfatase 1/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos de Insetos/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Microtúbulos/ultraestrutura , Oócitos/citologia , Oócitos/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Separase/genética , Separase/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Complexo Sinaptonêmico/metabolismo , Complexo Sinaptonêmico/ultraestrutura , Coesinas
10.
Dev Cell ; 46(3): 255-256, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30086299

RESUMO

Parental genomes are initially separate in the zygote following fertilization. A recent study in Science by Reichmann et al. (2018) reveals that dual spindles assemble around the two pronuclei in mouse embryos to maintain separation of the two parental genomes through the first zygotic division.


Assuntos
Núcleo Celular , Zigoto , Animais , Fertilização , Genoma , Camundongos
11.
G3 (Bethesda) ; 8(5): 1555-1569, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29514846

RESUMO

Bipolar spindle assembly occurs in the absence of centrosomes in the oocytes of most organisms. In the absence of centrosomes in Drosophila oocytes, we have proposed that the kinesin 6 Subito, a MKLP-2 homolog, is required for establishing spindle bipolarity and chromosome biorientation by assembling a robust central spindle during prometaphase I. Although the functions of the conserved motor domains of kinesins is well studied, less is known about the contribution of the poorly conserved N- and C- terminal domains to motor function. In this study, we have investigated the contribution of these domains to kinesin 6 functions in meiosis and early embryonic development. We found that the N-terminal domain has antagonistic elements that regulate localization of the motor to microtubules. Other parts of the N- and C-terminal domains are not required for microtubule localization but are required for motor function. Some of these elements of Subito are more important for either mitosis or meiosis, as revealed by separation-of-function mutants. One of the functions for both the N- and C-terminals domains is to restrict the CPC to the central spindle in a ring around the chromosomes. We also provide evidence that CDK1 phosphorylation of Subito regulates its activity associated with homolog bi-orientation. These results suggest the N- and C-terminal domains of Subito, while not required for localization to the central spindle microtubules, have important roles regulating Subito, by interacting with other spindle proteins and promoting activities such as bipolar spindle formation and homologous chromosome bi-orientation during meiosis.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Meiose , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Sequência Conservada , Análise Mutacional de DNA , Feminino , Metáfase , Microtúbulos/metabolismo , Mutação/genética , Oócitos/metabolismo , Fenótipo , Fosforilação , Domínios Proteicos , Transporte Proteico , Serina/metabolismo , Fuso Acromático/metabolismo , Transgenes
12.
Chromosoma ; 126(5): 595-604, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28791511

RESUMO

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.


Assuntos
Centrômero/genética , Epigênese Genética , Células Germinativas , Padrões de Herança , Animais , Centrômero/metabolismo , Proteína Centromérica A , Montagem e Desmontagem da Cromatina , Humanos
13.
Genetics ; 202(1): 61-75, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26564158

RESUMO

Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.


Assuntos
Segregação de Cromossomos , Cromossomos de Insetos/fisiologia , Proteínas de Drosophila/fisiologia , Meiose , Oócitos/citologia , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/fisiologia , Drosophila , Proteínas de Drosophila/farmacologia , Feminino , Proteínas Ativadoras de GTPase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Cinesinas/fisiologia , Masculino , Metáfase , Proteínas Associadas aos Microtúbulos/fisiologia , Mutagênese , Proteínas Serina-Treonina Quinases/fisiologia , Survivina , Proteínas rho de Ligação ao GTP/fisiologia
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