scholarly journals Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat

2005 ◽  
Vol 16 (4) ◽  
pp. 1800-1810 ◽  
Author(s):  
Nathaniel S. Edwards ◽  
Andrew W. Murray
Keyword(s):  
Dna Sequences ◽  
Cultured Cells ◽  
Protein A ◽  
Centromeric Dna ◽  
Eukaryotic Chromosome ◽  
Centromeric Repeat ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Dna Repeat ◽  
Egg Extracts

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.

scholarly journals Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

2015 ◽  
Vol 112 (11) ◽  
pp. E1263-E1271 ◽  
Author(s):  
Yalin Liu ◽  
Handong Su ◽  
Junling Pang ◽  
Zhi Gao ◽  
Xiu-Jie Wang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
B Chromosome ◽  
Chromosome 9 ◽  
Centromeric Dna ◽  
Repeat Sequences ◽  
Centromeric Repeat ◽  
And Function ◽  
Centromere Assembly ◽  
Derivatives Of

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

scholarly journals Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes

2013 ◽  
Vol 24 (7) ◽  
pp. 923-932 ◽  
Author(s):  
Dani L. Bodor ◽  
Luis P. Valente ◽  
João F. Mata ◽  
Ben E. Black ◽  
Lars E. T. Jansen
Keyword(s):  
Cell Cycle ◽  
Dna Sequences ◽  
Protein A ◽  
G1 Phase ◽  
Term Stability ◽  
Nucleosome Core ◽  
Long Term Stability ◽  
Centromere Position ◽  
Centromere Protein A

Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle–restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle–restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.

scholarly journals Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin

2007 ◽  
Vol 176 (6) ◽  
pp. 757-763 ◽  
Author(s):  
Paul S. Maddox ◽  
Francie Hyndman ◽  
Joost Monen ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Functional Genomics ◽  
Protein A ◽  
Common Mechanism ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Close Proximity ◽  
Single Protein ◽  
Histone H3 Variant ◽  
New Protein

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

scholarly journals Chromosome replication in cell-free systems from Xenopus eggs

1987 ◽  
Vol 317 (1187) ◽  
pp. 483-494 ◽  
Keyword(s):  
Cell Cycle ◽  
Dna Sequences ◽  
Chromosome Replication ◽  
Nucleosome Assembly ◽  
Cloning And Sequencing ◽  
Eukaryotic Chromosome ◽  
Initiation Of Replication ◽  
Egg Extracts ◽  
Acidic Proteins

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro . This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semiconservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986)). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro , only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.

scholarly journals Identification and characterization of centromeric sequences in Xenopus laevis

2020 ◽  
Author(s):  
Owen K Smith ◽  
Charles Limouse ◽  
Kelsey A Fryer ◽  
Nicole A Teran ◽  
Kousik Sundararajan ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Repetitive Sequences ◽  
Protein A ◽  
Repeat Sequences ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Egg Extracts ◽  
Genomic Studies

AbstractCentromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe

1988 ◽  
Vol 8 (2) ◽  
pp. 754-763
Author(s):  
B Fishel ◽  
H Amstutz ◽  
M Baum ◽  
J Carbon ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Sequences ◽  
Centromeric Dna ◽  
Centromere Function ◽  
Leu2 Gene ◽  
Centromeric Repeat ◽  
Sequence Elements ◽  
Chromosome Iii ◽  
Chromosome Ii

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.

scholarly journals Centromeric chromatin gets loaded

2007 ◽  
Vol 176 (6) ◽  
pp. 735-736 ◽  
Author(s):  
Christopher W. Carroll ◽  
Aaron F. Straight
Keyword(s):  
Chromosome Segregation ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Specific Loading ◽  
Histone H3 Variant

Centromeric nucleosomes contain a histone H3 variant called centromere protein A (CENP-A) that is required for kinetochore assembly and chromosome segregation. Two new studies, Jansen et al. (see p. 795 of this issue) and Maddox et al. (see p. 757 of this issue), address when CENP-A is deposited at centromeres during the cell division cycle and identify an evolutionally conserved protein required for CENP-A deposition. Together, these studies advance our understanding of centromeric chromatin assembly and provide a framework for investigating the molecular mechanisms that underlie the centromere-specific loading of CENP-A.

scholarly journals Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

1988 ◽  
Vol 8 (2) ◽  
pp. 754-763 ◽  
Author(s):  
B Fishel ◽  
H Amstutz ◽  
M Baum ◽  
J Carbon ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Sequences ◽  
Centromeric Dna ◽  
Centromere Function ◽  
Leu2 Gene ◽  
Centromeric Repeat ◽  
Sequence Elements ◽  
Chromosome Iii ◽  
Chromosome Ii

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.

scholarly journals The centromere-specific histone variant Cse4p (CENP-A) is essential for functional chromatin architecture at the yeast 2-μm circle partitioning locus and promotes equal plasmid segregation

2006 ◽  
Vol 174 (6) ◽  
pp. 779-790 ◽  
Author(s):  
Sujata Hajra ◽  
Santanu Kumar Ghosh ◽  
Makkuni Jayaram
Keyword(s):  
Chromosome Segregation ◽  
Protein A ◽  
Histone Variant ◽  
Centromeric Chromatin ◽  
Plasmid Segregation ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Spindle Disassembly ◽  
Late Telophase ◽  
2 Μm Plasmid

The centromere protein A homologue Cse4p is required for kinetochore assembly and faithful chromosome segregation in Saccharomyces cerevisiae. It has been regarded as the exquisite hallmark of centromeric chromatin. We demonstrate that Cse4 resides at the partitioning locus STB of the 2-μm plasmid. Cse4p-STB association is absolutely dependent on the plasmid partitioning proteins Rep1p and Rep2p and the integrity of the mitotic spindle. The kinetochore mutation ndc10-1 excludes Cse4p from centromeres without dislodging it from STB. Cse4p-STB association lasts from G1/S through late telophase during the cell cycle. The release of Cse4p from STB chromatin is likely mediated through spindle disassembly. A lack of functional Cse4p disrupts the remodeling of STB chromatin by the RSC2 complex, negates Rep2p binding and cohesin assembly at STB, and causes plasmid missegregation. Poaching of a specific histone variant by the plasmid to mark its partitioning locus with a centromere tag reveals yet another one of the molecular trickeries it performs for achieving chromosome- like fidelity in segregation.

scholarly journals Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors

2010 ◽  
Vol 190 (2) ◽  
pp. 177-185 ◽  
Author(s):  
Emily A. Bassett ◽  
Stacey Wood ◽  
Kevan J. Salimian ◽  
Sandya Ajith ◽  
Daniel R. Foltz ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Mitotic Spindle ◽  
Protein A ◽  
Centromeric Heterochromatin ◽  
Aurora B ◽  
Centromeric Chromatin ◽  
Chromosome Transmission ◽  
Centromere Protein A ◽  
Eukaryotic Species ◽  
Ubiquitous Presence

The nearly ubiquitous presence of repetitive centromere DNA sequences across eukaryotic species is in paradoxical contrast to their apparent functional dispensability. Centromeric chromatin is spatially delineated into the kinetochore-forming array of centromere protein A (CENP-A)–containing nucleosomes and the inner centromeric heterochromatin that lacks CENP-A but recruits the aurora B kinase that is necessary for correcting erroneous attachments to the mitotic spindle. We found that the self-perpetuating network of CENPs at the foundation of the kinetochore is intact at a human neocentromere lacking repetitive α-satellite DNA. However, aurora B is inappropriately silenced as a consequence of the altered geometry of the neocentromere, thereby compromising the error correction mechanism. This suggests a model wherein the neocentromere represents a primordial inheritance locus that requires subsequent generation of a robust inner centromere compartment to enhance fidelity of chromosome transmission.

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