scholarly journals N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

2020 ◽  
Vol 21 (17) ◽  
pp. 6175
Author(s):  
Hwei Ling Tan ◽  
Yi Bing Zeng ◽  
Ee Sin Chen
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Dna Sequences ◽  
Protein Turnover ◽  
Protein A ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Cell Extract ◽  
Insoluble Fraction ◽  
Independent Manner ◽  
Histone H3 Variant

Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.

scholarly journals Mis6/CENP-I maintains CENP-A nucleosomes against centromeric non-coding transcription during mitosis

2021 ◽  
Author(s):  
Hayato Hirai ◽  
Yuki Shogaki ◽  
Masamitsu Sato
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Underlying Mechanism ◽  
Core Region ◽  
The Core ◽  
Histone H3 Variant ◽  
Non Coding Rnas

Centromeres are established by nucleosomes containing the histone H3 variant CENP-A. CENP-A is recruited to centromeres by the Mis18-HJURP machinery. During mitosis, CENP-A recruitment ceases, implying the necessity of CENP-A maintenance at centromeres, although the exact underlying mechanism remains elusive. Herein, we show that the kinetochore protein Mis6 (CENP-I) retains CENP-A during mitosis in fission yeast. Eliminating Mis6 during mitosis caused immediate loss of pre-existing CENP-A at centromeres. CENP-A loss occurred due to the transcriptional upregulation of non-coding RNAs at the central core region of centromeres, as confirmed by the observation RNA polymerase II inhibition preventing CENP-A loss from centromeres in the mis6 mutant. Thus, we concluded that Mis6 blocks the indiscriminate transcription of non-coding RNAs at the core centromere, thereby retaining the epigenetic inheritance of CENP-A during mitosis.

scholarly journals snRNA and Heterochromatin Formation Are Involved in DNA Excision during Macronuclear Development in Stichotrichous Ciliates

2005 ◽  
Vol 4 (11) ◽  
pp. 1934-1941 ◽  
Author(s):  
Stefan A. Juranek ◽  
Sina Rupprecht ◽  
Jan Postberg ◽  
Hans J. Lipps
Keyword(s):  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
De Novo ◽  
Histone H3 ◽  
Heterochromatin Formation ◽  
Macronuclear Development ◽  
Dna Elimination ◽  
Histone H3 Variant ◽  
Specific Sequences

ABSTRACT Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant “X” is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

scholarly journals CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

2011 ◽  
Vol 195 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Valerie C. Coffman ◽  
Pengcheng Wu ◽  
Mark R. Parthun ◽  
Jian-Qiu Wu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Histone H3 ◽  
Kinetochore Assembly ◽  
Microtubule Attachment ◽  
Attachment Sites ◽  
Histone H3 Variant ◽  
Architectural Models

The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ∼40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1–DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.

scholarly journals Effective Potentials and Orbits in Weyl Conformastatic Slender Disk

2021 ◽  
Author(s):  
Paul Talbert ◽  
Steven Henikoff
Keyword(s):  
Cell Division ◽  
Dna Sequences ◽  
Histone H3 ◽  
Repeated Dna ◽  
Sequencing Technology ◽  
Repeated Dna Sequences ◽  
Effective Potentials ◽  
Histone H3 Variant ◽  
Long Read ◽  
Chromosomal Loci

Centromeres, the chromosomal loci where spindle fibers attach during cell division to segregate chromosomes, are typically found within satellite arrays in plants and animals. Satellite arrays have been difficult to analyze because they comprise megabases of tandem head-to-tail highly repeated DNA sequences. Much evidence suggests that centromeres are epigenetically defined by the location of nucleosomes containing the centromere-specific histone H3 variant cenH3, independently of the DNA sequences where they are located; however, the reason that cenH3 nucleosomes are generally found on rapidly evolving satellite arrays has remained unclear. Recently, long read sequencing technology has clarified the structures of satellite arrays and sparked rethinking of how they evolve, while new experiments and analyses have helped bring both understanding and further speculation about the role these highly repeated sequences play in centromere identification.

scholarly journals Enrichment of non-B-form DNA at D. melanogaster centromeres

2021 ◽  
Author(s):  
Venkata S. P. Patchigolla ◽  
Barbara G. Mellone
Keyword(s):  
Dna Sequences ◽  
Protein A ◽  
Secondary Structures ◽  
Eukaryotic Cell ◽  
Dna Melting ◽  
Dna Structures ◽  
Histone H3 Variant ◽  
Centromeric Protein ◽  
Dna Secondary Structures ◽  
Secondary Dna Structures

Centromeres are essential chromosomal regions that mediate the accurate inheritance of genetic information during eukaryotic cell division. Despite their conserved function, centromeres do not contain conserved DNA sequences and are instead epigenetically marked by the presence of the centromere-specific histone H3 variant CENP-A (centromeric protein A). The functional contribution of centromeric DNA sequences to centromere identity remains elusive. Previous work found that dyad symmetries with a propensity to adopt non-canonical secondary DNA structures are enriched at the centromeres of several species. These findings lead to the proposal that such non-canonical DNA secondary structures may contribute to centromere specification. Here, we analyze the predicted secondary structures of the recently identified centromere DNA sequences from Drosophila melanogaster. Although dyad symmetries are only enriched on the Y centromere, we find that other types of non-canonical DNA structures, including DNA melting and G-quadruplexes, are common features of all D. melanogaster centromeres. Our work is consistent with previous models suggesting that non-canonical DNA secondary structures may be conserved features of centromeres with possible implications for centromere specification.

scholarly journals Epigenetic inheritance of centromere identity in a heterologous system

2019 ◽  
Author(s):  
Virginie Roure ◽  
Bethan Medina-Pritchard ◽  
Eduard Anselm ◽  
A. Arockia Jeyaprakash ◽  
Patrick Heun
Keyword(s):  
Chromosome Segregation ◽  
Chromosomal Region ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Heterologous System ◽  
Histone H3 Variant ◽  
Self Association

SUMMARYThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

scholarly journals Trans-generational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

2020 ◽  
Author(s):  
Reinier F. Prosée ◽  
Joanna M. Wenda ◽  
Caroline Gabus ◽  
Kamila Delaney ◽  
Francoise Schwager ◽  
...  
Keyword(s):  
De Novo ◽  
Germ Line ◽  
Protein A ◽  
Histone H3 ◽  
Centromeric Chromatin ◽  
C Elegans ◽  
Centromere Function ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Meiosis I

AbstractCentromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is established de novo on chromatin during diplotene of meiosis I. Here we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but dispensable for centromere maintenance during embryogenesis. Worms homozygous for a CENP-A tail deletion maintain a functional centromere during development, but give rise to inviable offspring because they fail to re-establish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2, and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

scholarly journals Chromosomal variations of Lycoris species revealed by FISH with rDNAs and centromeric histone H3 variant associated DNAs

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0258028
Author(s):  
Mao-Sen Liu ◽  
Shih-Hsuan Tseng ◽  
Ching-Chi Tsai ◽  
Ting-Chu Chen ◽  
Mei-Chu Chung
Keyword(s):  
Dna Sequences ◽  
Histone H3 ◽  
Natural Populations ◽  
Centric Fusion ◽  
Distribution Patterns ◽  
Chromosomal Structure ◽  
Structure Changes ◽  
Cloning Method ◽  
Histone H3 Variant

Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species.

scholarly journals CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

2011 ◽  
Vol 194 (6) ◽  
pp. 855-871 ◽  
Author(s):  
Ben Moree ◽  
Corey B. Meyer ◽  
Colin J. Fuller ◽  
Aaron F. Straight
Keyword(s):  
Chromosome Segregation ◽  
Protein A ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Chromosomal Locus ◽  
C Protein ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Complex Protein

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

scholarly journals Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

2018 ◽  
Vol 29 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Shengya Cao ◽  
Keda Zhou ◽  
Zhening Zhang ◽  
Karolin Luger ◽  
Aaron F. Straight
Keyword(s):  
Protein A ◽  
Histone H3 ◽  
Intrinsic Property ◽  
Cell Cycles ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Differential Stability ◽  
The Stability

Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

Sign in / Sign up

Export Citation Format

Share Document