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Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis  [PDF]
Barbara G. Mellone ,Kathryn J. Grive equal contributor,Vladimir Shteyn equal contributor,Sarion R. Bowers,Isaac Oderberg,Gary H. Karpen
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002068
Abstract: Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.
Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State  [PDF]
Lisa Prendergast,Chelly van Vuuren,Agnieszka Kaczmarczyk,Volker Doering,Daniela Hellwig,Nadine Quinn,Christian Hoischen,Stephan Diekmann,Kevin F. Sullivan
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1001082
Abstract: Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.
Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State  [PDF]
Lisa Prendergast,Chelly van Vuuren,Agnieszka Kaczmarczyk,Volker Doering,Daniela Hellwig,Nadine Quinn,Christian Hoischen,Stephan Diekmann,Kevin F. Sullivan
PLOS Biology , 2011, DOI: 10.1371/journal.pbio.1001082
Abstract: Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.
Molecular cloning and sequence analysis of hamster CENP-A cDNA
Javier Figueroa, Carlos Pendón, Manuel M Valdivia
BMC Genomics , 2002, DOI: 10.1186/1471-2164-3-11
Abstract: We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein.We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural plasticity.Centromeres provide the essential functions for chromosome segregation. They act as regulators of mitosis and meiosis by controlling attachment of chromosomes to the spindle and regulating progression into anaphase [1-4]. In mammalian cells, the centromere is associated with large blocks of heterochromatin comprised of highly repetitive satellite DNA of unconserved sequence repeats among species. It have been hypothesized that centromere function is therefore not specified directly by DNA sequence, but rather by higher order DNA or chromatin structure [5]. Centromeric DNA and protein components have been suggested to evolve rapidly in evolution and be responsible for the organization of functional centromere of eme
Turning Meiosis into Mitosis  [PDF]
Isabelle d'Erfurth,Sylvie Jolivet,Nicole Froger,Olivier Catrice,Maria Novatchkova,Rapha?l Mercier
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000124
Abstract: Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis.
Turning Meiosis into Mitosis  [PDF]
Isabelle d'Erfurth,Sylvie Jolivet,Nicole Froger,Olivier Catrice,Maria Novatchkova,Rapha?l Mercier
PLOS Biology , 2009, DOI: 10.1371/journal.pbio.1000124
Abstract: Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis.
Molecular and evolutionary characteristics of the fraction of human alpha satellite DNA associated with CENP-A at the centromeres of chromosomes 1, 5, 19, and 21
Nathalie Pironon, Jacques Puechberty, Gérard Roizès
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-195
Abstract: In this paper, we show that the molecular mechanisms by which these arrays evolve are identical in multiple chromosomes: i) accumulation of crossovers that homogenise and expand the arrays into different domains and subdomains that are mostly unshared between homologues and ii) sporadic mutations and conversion events that simultaneously differentiate them from one another. Individual arrays are affected by these mechanisms to different extents that presumably increase with time. Repeats associated with CENP-A, where the centromere is formed, are subjected to the same evolutionary mechanisms, but constitute minor subsets that exhibit subtle sequence differences from those of the bulk repeats. While the DNA sequence per se is not essential for centromere localisation along an array, it appears that certain sequences can be selected against. On chromosomes 1 and 19, which are more affected by the above evolutionary mechanisms than are chromosomes 21 and 5, CENP-A associated repeats were also recovered from a second homogeneous array present on each chromosome. This could be a way for chromosomes to sustain mitosis and meiosis when the normal centromere locus is ineluctably undermined by the above mechanisms.We discuss, in light of these observations, possible scenarios for the normal evolutionary fates of human centromeric regions.Although human alpha satellite DNA sequences have been studied for decades, a number of their structural and evolutionary characteristics remain obscure. It is generally accepted that sequences constituting highly homogeneous arrays, including those within which the active centromere is formed, evolve in a concerted way [1]. In view of this concerted evolution, many authors have supposed that the repeats are homogenised with high efficiency, both intra-chromosomally and between homologues. At the same time, it has been shown that meiotic recombination is highly suppressed in the centromeric chromosomal regions [2-5]. Indeed, it was recently
Transgenerational Propagation and Quantitative Maintenance of Paternal Centromeres Depends on Cid/Cenp-A Presence in Drosophila Sperm  [PDF]
Nitika Raychaudhuri,Raphaelle Dubruille,Guillermo A. Orsi,Homayoun C. Bagheri,Benjamin Loppin,Christian F. Lehner
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1001434
Abstract: In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.
Sequential Assembly of Centromeric Proteins in Male Mouse Meiosis  [PDF]
María Teresa Parra ,Rocío Gómez,Alberto Viera,Elena Llano,Alberto M. Pendás,Julio S. Rufas,José A. Suja
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000417
Abstract: The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E.
A Role for the CAL1-Partner Modulo in Centromere Integrity and Accurate Chromosome Segregation in Drosophila  [PDF]
Chin-Chi Chen, Elizabeth Greene, Sarion R. Bowers, Barbara G. Mellone
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045094
Abstract: The relationship between the nucleolus and the centromere, although documented, remains one of the most elusive aspects of centromere assembly and maintenance. Here we identify the nucleolar protein, Modulo, in complex with CAL1, a factor essential for the centromeric deposition of the centromere-specific histone H3 variant, CID, in Drosophila. Notably, CAL1 localizes to both centromeres and the nucleolus. Depletion of Modulo, by RNAi, results in defective recruitment of newly-synthesized CAL1 at the centromere. Furthermore, depletion of Modulo negatively affects levels of CID at the centromere and results in chromosome missegregation. Interestingly, examination of Modulo localization during mitosis reveals it localizes to the chromosome periphery but not the centromere. Combined, the data suggest that rather than a direct regulatory role at the centromere, it is the nucleolar function of modulo which is regulating the assembly of the centromere by directing the localization of CAL1. We propose that a functional link between the nucleolus and centromere assembly exists in Drosophila, which is regulated by Modulo.
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