Topological similarity between the 2μm plasmid partitioning locus and the budding yeast centromere: evidence for a common evolutionary origin?

2013 ◽  
Vol 41 (2) ◽  
pp. 501-507 ◽  
Author(s):  
Makkuni Jayaram ◽  
Keng-Ming Chang ◽  
Chien-Hui Ma ◽  
Chu-Chun Huang ◽  
Yen-Ting Liu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Host Factors ◽  
Dependent Manner ◽  
Chromosomal Dna ◽  
Topological Similarity ◽  
Plasmid Partitioning ◽  
Histone H3 Variant ◽  
Functional States ◽  
Positive Supercoiling

The partitioning locus STB of the selfish plasmid, the 2μm circle, of Saccharomyces cerevisiae is essential for the propagation of this multi-copy extra-chromosomal DNA element with nearly chromosome-like stability. The functional competence of STB requires the plasmid-coded partitioning proteins Rep1 and Rep2 as well as host-coded proteins. Host factors that associate with STB in a Rep1- and Rep2-dependent manner also interact with centromeres, and play important roles in chromosome segregation. They include the cohesin complex and the centromere-specific histone H3 variant Cse4. The genetically defined point centromere of S. cerevisiae differs starkly from the much more widespread epigenetically specified regional centromeres of eukaryotes. The particularly small size of the S. cerevisiae centromere and the association of chromosome segregation factors with STB raise the possibility of an evolutionary link between these two partitioning loci. The unusual positive supercoiling harboured by the S. cerevisiae centromere and STB in vivo in their functional states, unveiled by recent experiments, bolsters the notion of their potential descent from an ancestral plasmid partitioning locus.

scholarly journals Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2

2000 ◽  
Vol 182 (21) ◽  
pp. 6014-6026 ◽  
Author(s):  
Thomas M. Rosche ◽  
Azeem Siddique ◽  
Michelle H. Larsen ◽  
David H. Figurski
Keyword(s):  
Binding Site ◽  
Broad Host Range ◽  
Dependent Manner ◽  
Chromosomal Dna ◽  
Obvious Effect ◽  
Partition System ◽  
Stable Maintenance ◽  
Plasmid Rk2

ABSTRACT Replication of the broad-host-range, IncPα plasmid RK2 requires two plasmid loci: trfA, the replication initiator gene, andoriV, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the incC gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for incC-mediated incompatibility is a KorB-binding site (OB). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the incC and korB genes individually has no obvious effect on Escherichia coli cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in anincC-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2.

scholarly journals CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

2019 ◽  
Author(s):  
N Cortes-Silva ◽  
J Ulmer ◽  
T Kiuchi ◽  
E Hsieh ◽  
G Cornilleau ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Gene Editing ◽  
Mitotic Progression ◽  
Critical Function ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Essential Function ◽  
Necessary And Sufficient ◽  
Assembly Pathways

AbstractAccurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

scholarly journals Cell cycle–dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast

2019 ◽  
Vol 30 (8) ◽  
pp. 1020-1036 ◽  
Author(s):  
Prashant K. Mishra ◽  
Gudjon Olafsson ◽  
Lars Boeckmann ◽  
Timothy J. Westlake ◽  
Ziad M. Jowhar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Growth Defects ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant ◽  
Cycle Dependent

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle–dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle–regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle–regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

scholarly journals The ATAD2/ANCCA homolog Yta7 cooperates with Scm3HJURP to deposit Cse4CENP-A at the centromere in yeast

2020 ◽  
Vol 117 (10) ◽  
pp. 5386-5393 ◽  
Author(s):  
Sara Shahnejat-Bushehri ◽  
Ann E. Ehrenhofer-Murray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Histone H3 ◽  
Nucleosome Assembly ◽  
Direct Role ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Assembly Factor

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP. The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.

scholarly journals CENP-H–containing Complex Facilitates Centromere Deposition of CENP-A in Cooperation with FACT and CHD1

2009 ◽  
Vol 20 (18) ◽  
pp. 3986-3995 ◽  
Author(s):  
Masahiro Okada ◽  
Katsuya Okawa ◽  
Toshiaki Isobe ◽  
Tatsuo Fukagawa
Keyword(s):  
Chromatin Remodeling ◽  
Mutant Cell ◽  
Histone H3 ◽  
Dependent Manner ◽  
Centromeric Chromatin ◽  
Conditional Mutant ◽  
Histone H3 Variant ◽  
A Subunit

Centromere identity is thought to be determined by epigenetic mechanisms. The centromere-specific histone H3 variant CENP-A plays a central role in specifying the locus where the centromere is constructed. However, the precise mechanisms that target CENP-A to centromeric chromatin are poorly understood. Here, we show that facilitates chromatin transcription (FACT) localizes to centromeres in a CENP-H–containing complex-dependent manner. In conditional mutant cell lines for SSRP1, a subunit of FACT, centromere targeting of newly synthesized CENP-A is severely inhibited. The chromatin remodeling factor CHD1 binds to SSRP1 both in vivo and in vitro and associates with centromeres. The centromeric localization of CHD1 is lost in SSRP1-depleted cells. RNA interference knockdown of CHD1 leads to a decrease in the amount of centromere localized CENP-A. These findings indicate that the CENP-H–containing complex facilitates deposition of newly synthesized CENP-A into centromeric chromatin in cooperation with FACT and CHD1.

scholarly journals Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (12) ◽  
pp. 2034-2044 ◽  
Author(s):  
Lars Boeckmann ◽  
Yoshimitsu Takahashi ◽  
Wei-Chun Au ◽  
Prashant K. Mishra ◽  
John S. Choy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Histone H3 ◽  
In Vitro Assays ◽  
Histone H3 Variant

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

scholarly journals Cdc7-mediated phosphorylation of Cse4 regulates high fidelity chromosome segregation in budding yeast

2021 ◽  
pp. mbc.E21-06-0323
Author(s):  
Prashant K. Mishra ◽  
Henry Wood ◽  
John Stanton ◽  
Wei-Chun Au ◽  
Jessica R. Eisenstatt ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Critical Role ◽  
High Fidelity ◽  
Dependent Manner ◽  
Growth Defects ◽  
A Cell ◽  
Cdc7 Kinase ◽  
Cycle Dependent

Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN) which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores and defects in chromosome segregation are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase dead variant of Cdc7 ( cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phosphodeficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.

scholarly journals Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination

2015 ◽  
Vol 26 (11) ◽  
pp. 2067-2079 ◽  
Author(s):  
Prashant K. Mishra ◽  
Jiasheng Guo ◽  
Lauren E. Dittman ◽  
Julian Haase ◽  
Elaine Yeh ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Chromosome Segregation ◽  
Histone H3 ◽  
Structure And Function ◽  
Epigenetic Mark ◽  
Critical Determinant ◽  
Essential Components ◽  
Histone H3 Variant ◽  
And Function

Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere ( CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.

scholarly journals FANCA Fine-Tunes Chromosome Segregation By Controlling BUBR1K250 Acetylation at the Kinetochores

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1040-1040
Author(s):  
Zahi Abdul Sater ◽  
Richa Sharma ◽  
Elizabeth Sierra Potchanant ◽  
Ying He ◽  
Grzegorz Nalepa
Keyword(s):  
Genomic Instability ◽  
Chromosome Segregation ◽  
Molecular Mechanisms ◽  
Spindle Assembly Checkpoint ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Cyclin B1 ◽  
Dependent Manner ◽  
Anaphase Onset

Abstract Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood. Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2. Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stable inheritance of CENP-A chromatin: Inner strength versus dynamic control

2020 ◽  
Vol 219 (10) ◽  
Author(s):  
Sreyoshi Mitra ◽  
Bharath Srinivasan ◽  
Lars E.T. Jansen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Dynamic Control ◽  
Centromeric Chromatin ◽  
Long Term Stability ◽  
Binding Partners ◽  
Histone H3 Variant ◽  
Long Lifetime

Chromosome segregation during cell division is driven by mitotic spindle attachment to the centromere region on each chromosome. Centromeres form a protein scaffold defined by chromatin featuring CENP-A, a conserved histone H3 variant, in a manner largely independent of local DNA cis elements. CENP-A nucleosomes fulfill two essential criteria to epigenetically identify the centromere. They undergo self-templated duplication to reestablish centromeric chromatin following DNA replication. More importantly, CENP-A incorporated into centromeric chromatin is stably transmitted through consecutive cell division cycles. CENP-A nucleosomes have unique structural properties and binding partners that potentially explain their long lifetime in vivo. However, rather than a static building block, centromeric chromatin is dynamically regulated throughout the cell cycle, indicating that CENP-A stability is also controlled by external factors. We discuss recent insights and identify the outstanding questions on how dynamic control of the long-term stability of CENP-A ensures epigenetic centromere inheritance.

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