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Benjamin Loppin - One of the best experts on this subject based on the ideXlab platform.
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Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus
PLoS Genetics, 2013Co-Authors: Guillermo A. Orsi, Pierre Couble, Ahmed Algazeery, Michèle Capri, Laure Sapey-triomphe, Béatrice Horard, Henri Gruffat, Ounissa Aït-ahmed, Régis E. Meyer, Benjamin LoppinAbstract:The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male Pronucleus. yem loss of function alleles affect male Pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male Pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.
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Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus
PLoS Genetics, 2013Co-Authors: Guillermo Orsi, Pierre Couble, Ahmed Algazeery, Regis Meyer, Michèle Capri, Laure Sapey-triomphe, Béatrice Horard, Henri Gruffat, Ounissa Aït-ahmed, Benjamin LoppinAbstract:The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male Pronucleus. yem loss of function alleles affect male Pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male Pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation. Citation: Orsi GA, Algazeery A, Meyer RE, Capri M, Sapey-Triomphe LM, et al. (2013) Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus. PLoS Genet 9(2): e1003285.
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Wolbachia-mediated cytoplasmic incompatibility is associated with impaired histone deposition in the male Pronucleus.
PLoS Pathogens, 2009Co-Authors: Frédéric Landmann, Guillermo A. Orsi, Benjamin Loppin, William SullivanAbstract:Wolbachia is a bacteria endosymbiont that rapidly infects insect populations through a mechanism known as cytoplasmic incompatibility (CI). In CI, crosses between Wolbachia-infected males and uninfected females produce severe cell cycle defects in the male Pronucleus resulting in early embryonic lethality. In contrast, viable progeny are produced when both parents are infected (the Rescue cross). An important consequence of CI-Rescue is that infected females have a selective advantage over uninfected females facilitating the rapid spread of Wolbachia through insect populations. CI disrupts a number of prophase and metaphase events in the male Pronucleus, including Cdk1 activation, chromosome condensation, and segregation. Here, we demonstrate that CI disrupts earlier interphase cell cycle events. Specifically, CI delays the H3.3 and H4 deposition that occurs immediately after protamine removal from the male Pronucleus. In addition, we find prolonged retention of the replication factor PCNA in the male Pronucleus into metaphase, indicating progression into mitosis with incompletely replicated DNA. We propose that these CI-induced interphase defects in de novo nucleosome assembly and replication are the cause of the observed mitotic condensation and segregation defects. In addition, these interphase chromosome defects likely activate S-phase checkpoints, accounting for the previously described delays in Cdk1 activation. These results have important implications for the mechanism of Rescue and other Wolbachia-induced phenotypes.
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The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization.
PLoS Genetics, 2007Co-Authors: Emilie Bonnefoy, Guillermo A. Orsi, Pierre Couble, Benjamin LoppinAbstract:In many animal species, the sperm DNA is packaged with male germ line--specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male Pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male Pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male Pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.
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HIRA, une molécule de l’oeuf qui contrôle la formation du Pronucleus mâle
M S-medecine Sciences, 2006Co-Authors: Benjamin Loppin, Pierre CoubleAbstract:> L’architecture du noyau eucaryote est extremement variable selon le type cellulaire. La structure intime de la chromatine est en revanche remarquablement conservee a l’echelle de son unite fonctionnelle, le nucleosome, particule resultant de l’assemblage des quatre types d’histones (H2A, H2B, H3 et H4) et de la molecule d’ADN. Chez de nombreuses especes, le noyau du spermatozoide deroge a cette regle fondamentale en adoptant une organisation de sa chromatine radicalement differente. Selon les especes, les histones sont totalement ou partiellement remplacees dans le noyau du gamete mâle par des petites proteines chromosomiques tres basiques appelees protamines. Les protamines permettent a l’ADN d’atteindre un niveau de compaction tres eleve. A la fecondation, lorsque le noyau du spermatozoide est libere dans le cytoplasme de l’œuf, la chromatine paternelle doit rapidement eliminer ses protamines pour reacquerir une organisation en nucleosomes essentielle pour le developpement de l’embryon. Nous avons identifie une mutation chez la drosophile, appelee sesame, qui affecte tres specifiquement ce processus [1]. Dans les œufs pondus par des femelles mutantes, le noyau du spermatozoide provenant d’un mâle sauvage est incapable de decondenser sa chromatine. Le Pronucleus mâle, anormalement condense, est alors exclu de la premiere mitose zygotique et les embryons ne se developpent qu’avec le seul jeu de chromosomes maternels. Nous avons recemment decouvert que la mutation sesame affecte le gene Hira qui code un facteur d’assemblage de la chromatine present chez tous les eucaryotes [2]. La mutation induit le remplacement d’un seul residu tres conserve, dans un domaine de HIRA predit pour interagir avec d’autres proteines. Les proprietes d’assemblage de la chromatine du facteur HIRA ont ete recemment caracterisees in vitro par le groupe de Genevieve Almouzni et ses collaborateurs [3]. HIRA fait ainsi partie d’un complexe proteique capable d’assembler des nucleosomes independamment de la synthese d’ADN, par opposition au complexe CAF-1, dont la fonction est d’assembler les nucleosomes sur l’ADN en cours de replication ou de reparation. Dans un œuf sauvage de drosophile, le noyau du spermatozoide doit assembler sa chromatine en remplacant ses protamines par des histones qui sont fournies par l’œuf. Ce processus, qui s’accompagne de la decondensation de la chromatine paternelle, intervient bien avant que le Pronucleus mâle ne replique son ADN, ce qu’il fait au moment ou il rejoint son partenaire femelle. La formation du Pronucleus mâle releve donc d’un mode d’assemblage independant de la HIRA, une molecule de l’œuf qui controle la formation du Pronucleus mâle
Pierre Couble - One of the best experts on this subject based on the ideXlab platform.
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Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus
PLoS Genetics, 2013Co-Authors: Guillermo A. Orsi, Pierre Couble, Ahmed Algazeery, Michèle Capri, Laure Sapey-triomphe, Béatrice Horard, Henri Gruffat, Ounissa Aït-ahmed, Régis E. Meyer, Benjamin LoppinAbstract:The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male Pronucleus. yem loss of function alleles affect male Pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male Pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.
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Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus
PLoS Genetics, 2013Co-Authors: Guillermo Orsi, Pierre Couble, Ahmed Algazeery, Regis Meyer, Michèle Capri, Laure Sapey-triomphe, Béatrice Horard, Henri Gruffat, Ounissa Aït-ahmed, Benjamin LoppinAbstract:The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male Pronucleus. yem loss of function alleles affect male Pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male Pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation. Citation: Orsi GA, Algazeery A, Meyer RE, Capri M, Sapey-Triomphe LM, et al. (2013) Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus. PLoS Genet 9(2): e1003285.
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The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization.
PLoS Genetics, 2007Co-Authors: Emilie Bonnefoy, Guillermo A. Orsi, Pierre Couble, Benjamin LoppinAbstract:In many animal species, the sperm DNA is packaged with male germ line--specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male Pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male Pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male Pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.
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HIRA, une molécule de l’oeuf qui contrôle la formation du Pronucleus mâle
M S-medecine Sciences, 2006Co-Authors: Benjamin Loppin, Pierre CoubleAbstract:> L’architecture du noyau eucaryote est extremement variable selon le type cellulaire. La structure intime de la chromatine est en revanche remarquablement conservee a l’echelle de son unite fonctionnelle, le nucleosome, particule resultant de l’assemblage des quatre types d’histones (H2A, H2B, H3 et H4) et de la molecule d’ADN. Chez de nombreuses especes, le noyau du spermatozoide deroge a cette regle fondamentale en adoptant une organisation de sa chromatine radicalement differente. Selon les especes, les histones sont totalement ou partiellement remplacees dans le noyau du gamete mâle par des petites proteines chromosomiques tres basiques appelees protamines. Les protamines permettent a l’ADN d’atteindre un niveau de compaction tres eleve. A la fecondation, lorsque le noyau du spermatozoide est libere dans le cytoplasme de l’œuf, la chromatine paternelle doit rapidement eliminer ses protamines pour reacquerir une organisation en nucleosomes essentielle pour le developpement de l’embryon. Nous avons identifie une mutation chez la drosophile, appelee sesame, qui affecte tres specifiquement ce processus [1]. Dans les œufs pondus par des femelles mutantes, le noyau du spermatozoide provenant d’un mâle sauvage est incapable de decondenser sa chromatine. Le Pronucleus mâle, anormalement condense, est alors exclu de la premiere mitose zygotique et les embryons ne se developpent qu’avec le seul jeu de chromosomes maternels. Nous avons recemment decouvert que la mutation sesame affecte le gene Hira qui code un facteur d’assemblage de la chromatine present chez tous les eucaryotes [2]. La mutation induit le remplacement d’un seul residu tres conserve, dans un domaine de HIRA predit pour interagir avec d’autres proteines. Les proprietes d’assemblage de la chromatine du facteur HIRA ont ete recemment caracterisees in vitro par le groupe de Genevieve Almouzni et ses collaborateurs [3]. HIRA fait ainsi partie d’un complexe proteique capable d’assembler des nucleosomes independamment de la synthese d’ADN, par opposition au complexe CAF-1, dont la fonction est d’assembler les nucleosomes sur l’ADN en cours de replication ou de reparation. Dans un œuf sauvage de drosophile, le noyau du spermatozoide doit assembler sa chromatine en remplacant ses protamines par des histones qui sont fournies par l’œuf. Ce processus, qui s’accompagne de la decondensation de la chromatine paternelle, intervient bien avant que le Pronucleus mâle ne replique son ADN, ce qu’il fait au moment ou il rejoint son partenaire femelle. La formation du Pronucleus mâle releve donc d’un mode d’assemblage independant de la HIRA, une molecule de l’œuf qui controle la formation du Pronucleus mâle
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hira une molecule de l oeuf qui controle la formation du Pronucleus mâle
M S-medecine Sciences, 2006Co-Authors: Benjamin Loppin, Pierre CoubleAbstract:> L’architecture du noyau eucaryote est extremement variable selon le type cellulaire. La structure intime de la chromatine est en revanche remarquablement conservee a l’echelle de son unite fonctionnelle, le nucleosome, particule resultant de l’assemblage des quatre types d’histones (H2A, H2B, H3 et H4) et de la molecule d’ADN. Chez de nombreuses especes, le noyau du spermatozoide deroge a cette regle fondamentale en adoptant une organisation de sa chromatine radicalement differente. Selon les especes, les histones sont totalement ou partiellement remplacees dans le noyau du gamete mâle par des petites proteines chromosomiques tres basiques appelees protamines. Les protamines permettent a l’ADN d’atteindre un niveau de compaction tres eleve. A la fecondation, lorsque le noyau du spermatozoide est libere dans le cytoplasme de l’œuf, la chromatine paternelle doit rapidement eliminer ses protamines pour reacquerir une organisation en nucleosomes essentielle pour le developpement de l’embryon. Nous avons identifie une mutation chez la drosophile, appelee sesame, qui affecte tres specifiquement ce processus [1]. Dans les œufs pondus par des femelles mutantes, le noyau du spermatozoide provenant d’un mâle sauvage est incapable de decondenser sa chromatine. Le Pronucleus mâle, anormalement condense, est alors exclu de la premiere mitose zygotique et les embryons ne se developpent qu’avec le seul jeu de chromosomes maternels. Nous avons recemment decouvert que la mutation sesame affecte le gene Hira qui code un facteur d’assemblage de la chromatine present chez tous les eucaryotes [2]. La mutation induit le remplacement d’un seul residu tres conserve, dans un domaine de HIRA predit pour interagir avec d’autres proteines. Les proprietes d’assemblage de la chromatine du facteur HIRA ont ete recemment caracterisees in vitro par le groupe de Genevieve Almouzni et ses collaborateurs [3]. HIRA fait ainsi partie d’un complexe proteique capable d’assembler des nucleosomes independamment de la synthese d’ADN, par opposition au complexe CAF-1, dont la fonction est d’assembler les nucleosomes sur l’ADN en cours de replication ou de reparation. Dans un œuf sauvage de drosophile, le noyau du spermatozoide doit assembler sa chromatine en remplacant ses protamines par des histones qui sont fournies par l’œuf. Ce processus, qui s’accompagne de la decondensation de la chromatine paternelle, intervient bien avant que le Pronucleus mâle ne replique son ADN, ce qu’il fait au moment ou il rejoint son partenaire femelle. La formation du Pronucleus mâle releve donc d’un mode d’assemblage independant de la HIRA, une molecule de l’œuf qui controle la formation du Pronucleus mâle
Yong-kook Kang - One of the best experts on this subject based on the ideXlab platform.
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gradual development of a genome wide h3 k9 trimethylation pattern in paternally derived pig Pronucleus
Developmental Dynamics, 2007Co-Authors: Young-sun Jeong, Seungeun Yeo, Jung-sun Park, Kyung-kwang Lee, Yong-kook KangAbstract:The cytoplasm of a mature oocyte contains many protein complexes that are programmed to restructure incoming sperm chromatins on fertilization. Of the complicated biochemical events that these functional machineries control, the most impressive and important is epigenetic reprogramming. Despite its importance in epigenetic resetting, or “de-differentiation,” of gamete genomes back to an incipient status, the mechanisms of epigenetic reprogramming do not seem to be conserved among mammals. Here, we report that, unlike in the mouse, the pig sperm-derived Pronucleus is markedly trimethylated at lysine 9 of histone H3 (H3-m3K9), which might be associated with preservation of paternally derived cytosine methylation in pig zygotes. The male H3-m3K9 pattern is gradually established during Pronucleus development, and this process occurs independently of DNA replication. Considering these unique epigenetic features, the pig zygote is, we believe, suited to serve as another model of epigenetic reprogramming that is antithetical to the well-characterized mouse model. Developmental Dynamics 236:1509–1516, 2007. © 2007 Wiley-Liss, Inc.
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DNA methylation state is preserved in the sperm-derived Pronucleus of the pig zygote.
The International journal of developmental biology, 2007Co-Authors: Young-sun Jeong, Seungeun Yeo, Jung-sun Park, Deog-bon Koo, Won-kyung Chang, Kyung-kwang Lee, Yong-kook KangAbstract:DNA methylation reprogramming (DMR) during preimplantation development erases differentiation-associated, unessential epigenetic information accumulated during gametogenesis, and ultimately brings pluripotency to the resulting embryo. Two patterns of DMR of sperm-derived Pronucleus have been reported in mammals. In the first, the male Pronucleus is actively demethylated whereas in the second, the methylation state seems to be maintained. The maintenance-type DMR has been seen only through immunocytochemical observations, and waits to be proven by additional molecular-level evidence. We demonstrate that, in pig, paternally derived DNA methylation is preserved during Pronucleus development, based on the following observations. First, immunostaining of pig zygotes at different time points showed the DNA methylation state to be balanced between parental pronuclei throughout Pronucleus development. Second, bisulfite analysis of PRE-1 repetitive sequences found mono- and polyspermic eggs to have similar methylation states. Third, the methylation state of a human erythropoietin gene delivered by transgenic pig spermatozoa was maintained in the male Pronucleus. Finally, 5-aza-2'-deoxycytidine treatment, which blocks re-methylation, did not show the male Pronucleus to be stalled in a demethylated state. In pig zygotes, paternally derived cytosine methylation was preserved throughout Pronucleus development. These findings from multilateral DMR analyses provide further support to the view that DMR occurs in a non-conserved manner during early mammalian development.
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Gradual development of a genome-wide H3-K9 trimethylation pattern in paternally derived pig Pronucleus.
Developmental dynamics : an official publication of the American Association of Anatomists, 2007Co-Authors: Young-sun Jeong, Seungeun Yeo, Jung-sun Park, Kyung-kwang Lee, Yong-kook KangAbstract:The cytoplasm of a mature oocyte contains many protein complexes that are programmed to restructure incoming sperm chromatins on fertilization. Of the complicated biochemical events that these functional machineries control, the most impressive and important is epigenetic reprogramming. Despite its importance in epigenetic resetting, or "de-differentiation," of gamete genomes back to an incipient status, the mechanisms of epigenetic reprogramming do not seem to be conserved among mammals. Here, we report that, unlike in the mouse, the pig sperm-derived Pronucleus is markedly trimethylated at lysine 9 of histone H3 (H3-m(3)K9), which might be associated with preservation of paternally derived cytosine methylation in pig zygotes. The male H3-m(3)K9 pattern is gradually established during Pronucleus development, and this process occurs independently of DNA replication. Considering these unique epigenetic features, the pig zygote is, we believe, suited to serve as another model of epigenetic reprogramming that is antithetical to the well-characterized mouse model.
Maria Imschenetzky - One of the best experts on this subject based on the ideXlab platform.
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During male pronuclei formation chromatin remodeling is uncoupled from nucleus decondensation.
Journal of Cellular Biochemistry, 2005Co-Authors: Antonia Monardes, Claudio Iribarren, Violeta Morin, Paula Bustos, Marcia Puchi, Maria ImschenetzkyAbstract:Male Pronucleus formation involves sperm nucleus decondensation and sperm chromatin remodeling. In sea urchins, male Pronucleus decondensation was shown to be modulated by protein kinase C and a cdc2-like kinase sensitive to olomoucine in vitro assays. It was further demonstrated that olomoucine blocks SpH2B and SpH1 phosphorylation. These phosphorylations were postulated to participate in the initial steps of male chromatin remodeling during male Pronucleus formation. At final steps of male chromatin remodeling, all sperm histones (SpH) disappear from male chromatin and are subsequently degraded by a cysteine protease. As a result of this remodeling, the SpH are replaced by maternal histone variants (CS). To define if sperm nucleus decondensation is coupled with sperm chromatin remodeling, we have followed the loss of SpH in zygotes treated with olomoucine. SpH degradation was followed with anti-SpH antibodies that had no cross-reactivity with CS histone variants. We found that olomoucine blocks SpH1 and SpH2B phosphorylation and inhibits male Pronucleus decondensation in vivo. Interestingly, the normal schedule of SpH degradation remains unaltered in the presence of olomoucine. Taken together these results, it was concluded that male nucleus decondensation is uncoupled from the degradation of SpH associated to male chromatin remodeling. From these results, it also emerges that the phosphorylation of SpH2B and SpH1 is not required for the degradation of the SpH that is concurrent to male chromatin remodeling. © 2005 Wiley-Liss, Inc.
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Hybrid nucleoprotein particles containing a subset of male and female histone variants form during male Pronucleus formation in sea urchins.
Journal of cellular biochemistry, 1996Co-Authors: Maria Imschenetzky, Violeta Morin, María Isabel Oliver, Soraya E. Gutierrez, Cecilia Cárdenas Garrido, Alejandra Bustos, Marcia PuchiAbstract:To determine the changes in chromatin organization during male Pronucleus remodeling, we have compared the composition of nucleoprotein particles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated from sea urchin gametes and zygotes containing partially decondensed (15 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) male Pronucleus and digested with nucleases. The NP-ps generated were analyzed in agarose gels, and their histone composition was determined. Sperm core histones (SpH) and cleavage stage (CS) variants were identified by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, which migrated in agarose gels between DNA fragments of 1.78-1.26 Kb. Sperm chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type of NP-ps was obtained by digestion of unfertilized egg nuclei, either with ENase or MNase; the NP-ps was located in the region of the agarose gel corresponding to DNA fragments of 3.4-1.95 Kb [Imschenetzky et al. (1989): Exp Cell Res 182:436-444]. When whole nuclei from zygotes containing the female Pronucleus and a partially remodeled male Pronucleus were digested with ENase, a single NP-ps was generated, which migrated between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested with MNase, two NP-ps were generated; the slower migrating NP-ps (s) was located in the same position of the agarose gel as those resulting from ENase digestion and the faster migrating NP-ps (f) migrated between DNA fragments of 1.95-1.26 Kb. It was found that NP-ps (s) contained only CS histone variants, whereas NP-ps (f) were formed by a subset of SpH and by CS histone variants. When nuclei from zygotes containing a fully decondensed male Pronucleus were digested either with ENase or MNase, a single type of NP-ps was observed, which migrated in the same position as NP-ps (s) in agarose gels. This particle contained only CS histone variants. On the basis of the histone compositions and on electrophoretic similarities, it was concluded that NP-ps (s) originated from the female Pronucleus and that NP-ps (f) were generated from the partially remodeled male Pronucleus. Consequently, our results indicate that at an intermediate stage of male Pronucleus remodeling the chromatin is formed by NP-ps containing a subset of both SpH and of CS histone variants, whereas at final stages of male Pronucleus decondensation chromatin organization is similar to that of the female Pronucleus.
Philippe Collas - One of the best experts on this subject based on the ideXlab platform.
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Formation of the sea urchin male Pronucleus in cell-free extracts.
Molecular Reproduction and Development, 2000Co-Authors: Philippe CollasAbstract:At fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional male Pronucleus. We have investigated the formation of the male Pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear transformation observed in vitro is the disassembly of the sperm nuclear lamina as a result of lamin B phosphorylation mediated by egg protein kinase C. The conical sperm nucleus then decondenses into a spherical Pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles with one another. Three cytoplasmic vesicle fractions with distinct properties are required for the formation of the male pronuclear envelope. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the NE. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus, formation of the male pronuclear envelope involves a highly ordered series of reactions.
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Remodeling the sperm nucleus into a male Pronucleus at fertilization.
Theriogenology, 1998Co-Authors: Philippe Collas, Dominic PocciaAbstract:After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male Pronucleus. We have investigated the formation of the male Pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical Pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.
