Fungi as models of centromere innovation: from DNA sequence to 3-dimensional arrangement.

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.

Centromeres are specialized chromosomal domains involved in kinetochore formation and faithful chromosome segregation. Despite a high level of functional conservation, centromeres are not identified by DNA sequences, but by epigenetic means. Universally, centromeres are typically formed on highly repetitive DNA, which were previously considered to be silent. However, recent studies have shown that transcription occurs in this region, known as centromeric-derived RNAs (cenRNAs). CenRNAs that contribute to fundamental aspects of centromere function have been recently investigated in detail. However, the distribution, behavior and contributions of centromeric transcripts are still poorly understood. The aim of this article is to provide an overview of the roles of cenRNAs in centromere formation and function. We describe the structure and DNA sequence of centromere from yeast to human. In addition, we briefly introduce the roles of cenRNAs in centromere formation and function, kinetochore structure, accurate chromosome segregation, and pericentromeric heterochromatin assembly. Centromeric circular RNAs (circRNAs) and R-loops are rising stars in centromere function. CircRNAs have been successfully identified in various species with the assistance of high-throughput sequencing and novel computational approaches for non-polyadenylated RNA transcripts. Centromeric R-loops can be identified by the single-strand DNA ligation-based library preparation technique. But the molecular features and function of these centromeric R-loops and circRNAs are still being investigated. In this review, we summarize recent findings on the epigenetic regulation of cenRNAs across species, which would provide useful information about cenRNAs and interesting hints for further studies.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

We observed that a YCp-type vector having the centromeric DNA (CEN) sequence previously isolated from the genome, but not a YRp-type vector lacking the CEN sequence, induced pseudohyphal growth in a dimorphic fungi, Candida maltosa, which had been shown to be closely related to Candida albicans by phylogenetic analysis. Deletion analysis of the CEN sequence revealed that the intact CEN sequence was not required for the induction, but part of it, having partial centromeric activity, was enough for the induction. By screening the gene library of this yeast for the sequences which induced pseudohyphal growth, we isolated three different DNA fragments which also had part of the centromere-like sequence. Partial centromeric activity of these fragments was confirmed by three criteria: low copy number and high stability of the plasmids carrying these fragments and rearrangement at high frequency of the plasmid DNA with one of these fragments plus the CEN sequence. Furthermore, when the GGTAGCG sequence commonly found in one copy in each of these four sequences was mutated in the CEN sequence by site-directed mutagenesis, both partial centromeric activity and pseudohyphal growth-inducing activity of the CEN sequence were lost. These results indicated that part of CEN region with partial centromeric activity induces pseudohyphal growth in C. maltosa. It is suggested that some cellular components which interact with the sequence containing GGTAGCG required for centromeric activity are involved in the regulation of the transition between yeast forms and pseudohyphal forms of the cells.

Proper segregation of chromosomes is an essential component of cell division. The centromere is the locus at which the kinetochore-the proteinaceous complex that ties chromosomes to microtubules-forms during mitosis and meiosis. Thus, the centromere is critical for equal segregation of chromosomes. The centromere is characterized by both protein and DNA elements: the histone H3 variant CENP-A epigenetically defines the location of the centromere while centromeric DNA sequences are neither necessary nor sufficient for centromere function. Paradoxically, the DNA sequences play a critical role in new centromere formation. In this essay, we discuss the contribution of both epigenetics and genetics at the centromere. Understanding these contributions is vital to efforts to control centromere formation on synthetic/artificial chromosomes and centromere strength on natural ones.

ABSTRACTCentromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1 in vitro, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation, and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans. This article has an associated First Person interview with the first author of the paper.

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

The kinetochore is a specialized structure composed of centromeric DNA and a large number of proteins. The primary function of the kinetochore is to connect chromosomes with the mitotic spindle throughout the cell cycle and to monitor the fidelity of these attachments in order to ensure proper chromosome segregation. Despite the fact that chromosome segregation is directed by the kinetochore, the architecture and assembly of such an intricate structure remains elusive. We use budding yeast as a model system to characterize direct interactions among the central domain of the kinetochore proteins, specifically the COMA-complex. The COMA-complex consists of four proteins; two nonessential proteins Ctf19 and Mcm21 and two essential proteins Ame1 and Okp1. Although the chromosome segregation is a highly conserved process, the human orthologues of the two essential Ame1 and Okp1 proteins have not been identified. The tetrameric complex is the core of the COMA-network which is composed of seven additional nonessential proteins: Ctf3, Mcm16, Mcm22, Chl4, Iml3, Nkp1 and Nkp2, more loosely associated. According to the central localization within the kinetochore, the COMA-complex represents one of the linker complexes (together with the Mtw1-, Ndc80- and Spc105-complexes) bridging the centromere-associate inner proteins with microtubule-bounded outer kinetochore proteins. Here we present a biochemical approach to reconstitute and to characterize the budding yeast COMA-network. Our first aim was to reconstitute the COMA-complex in vitro and in vivo. A stabile heterodimer consisting of Ctf19 and Mcm21 proteins could be reconstituted as a tetrameric complex in solution. The Ame1 and Okp1 heterodimer showed noticeable instability and we were not able to reconstitute it. Surprisingly, the trimeric Ctf19, Mcm21 and Okp1-complex could be assembled in vivo independently of the Ame1 essential protein. Moreover, we demonstrated that the Okp1 coiled-coil region per se is sufficient to form a complex with Ctf19 and Mcm21 proteins in vitro. The tetrameric COMA-complex could have also been reconstituted in vitro, but the amount and the stoichiometry of the components were not satisfactory. In solution, the COMA-complex showed oligomerization behavior. Through the protein purification experiments, we also found that the Ctf19, Mcm21 and Okp1-complex as well as the Ame1 protein separately may bind to unspecific, E.coli RNA. To determine other kinetochore subunits that can directly or indirectly associate with the COMA-components, we performed co-immunoprecipitation from yeast cells with either Okp1-TAP or Ame1-TAP tagged proteins. Among many known interacting partners, the Dsn1 component of the Mtw1-central kinetochore complex was identified. To support this finding and to test if the binding between the Dsn1 and the COMA-proteins is direct or indirect, we performed in vitro reticulocyte lysate binding assay. The interaction between the Mtw1- and the COMA-complexes via the Dsn1 protein was confirmed. Additional information has been gained from the co-immunoprecipitation experiments using budding yeast cells. We identified two proteins from the COMA-network, Nkp1 and Nkp2 proteins, as highly enriched. Since this may reflect the close proximity of these two proteins to the core of the COMA-network, we purified separately Nkp1 and Nkp2 dimer (which revealed the stabile heterodimer formation between these two Nkp proteins), combined it with the recombinant COMA-complex and reconstituted the hexameric protein complex at a 1:1:1:1:1:1 stoichiometry. Taken together, this study led to proposal of a new model for the spatial organization of the COMA-network. In summary, we used affinity based protein isolation to identify new direct binding partners within the central domain of the budding yeast kinetochore. Our findings improve the current understanding of the overall kinetochore architecture. The complete characterization of the kinetochore structure and organization has to be fully known, ultimately leading to three-dimensional vision and biochemical features of the kinetochore complexes, in order to unravel the mechanisms of chromosome segregation and maintenance of genome stability. Our work is therefore one step further in answering relevant biological and medical questions concerning faithful chromosome segregation during mitosis.

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis.

The centromere is essential for faithful chromosome segregation by providing the site for kinetochore assembly. Although the role of the centromere is conserved throughout evolution, the DNA sequences associated with centromere regions are highly divergent among species and it remains to be determined how centromere DNA directs kinetochore formation. Despite the active use of chicken DT40 cells in studies of chromosome segregation, the sequence of the chicken centromere was unclear. Here, we performed a comprehensive analysis of chicken centromere DNA which revealed unique features of chicken centromeres compared with previously studied vertebrates. Centromere DNA sequences from the chicken macrochromosomes, with the exception of chromosome 5, contain chromosome-specific homogenous tandem repetitive arrays that span several hundred kilobases. In contrast, the centromeres of chromosomes 5, 27, and Z do not contain tandem repetitive sequences and span non-tandem-repetitive sequences of only approximately 30 kb. To test the function of these centromere sequences, we conditionally removed the centromere from the Z chromosome using genetic engineering and have shown that that the non-tandem-repeat sequence of chromosome Z is a functional centromere.

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.

Despite their common function, centromeric DNA sequences are not conserved between organisms. Most centromeres of animals and plants so far investigated have now been shown to consist of large blocks of tandemly repeated satellite sequences that are embedded in recombination-deficient heterochromatic regions. This central domain of satellite sequences that is postulated to mediate spindle attachment is surrounded by pericentromeric sequences incorporating various classes of repetitive sequences often including retroelements. The centromeric satellite DNA sequences are amongst the most rapidly evolving sequences and pose some fundamental problems of maintaining function. In this overview, we will discuss work on centromeric repetitive sequences in Arabidopsis thaliana and its relatives, and highlight some of the common features that are emerging when analysing closely related species.