The overexpression of a Saccharomyces cerevisiae centromeric histone H3 variant mutant protein leads to a defect in kinetochore biorientation

Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA and mediate attachment to the mitotic spindle. Because centromeric sequences are not conserved, centromere identity is propagated by an epigenetic mechanism. All eukaryotes contain an essential histone H3 variant (CenH3) that localizes exclusively to centromeres. Because CenH3 is required for kinetochore assembly and is likely to be the epigenetic mark that specifies centromere identity, it is critical to elucidate the mechanisms that assemble and maintain CenH3 exclusively at centromeres. To learn more about the functions and regulation of CenH3, we isolated mutants in the budding yeast CenH3 that are lethal when overexpressed. These CenH3 mutants fall into three unique classes: (I) those that localize to euchromatin but do not alter kinetochore function, (II) those that localize to the centromere and disrupt kinetochore function, and (III) those that no longer target to the centromere but still disrupt chromosome segregation. We found that a class III mutant is specifically defective in the ability of sister kinetochores to biorient and attach to microtubules from opposite spindle poles, indicating that CenH3 mutants defective in kinetochore biorientation can be obtained.     

Stretching the rules: monocentric chromosomes with multiple centromere domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.     

Searching for a Common Centromeric Structural Motif: Drosophila Centromeric Satellite DNAs Show Propensity to form Telomeric-Like Unusual DNA Structures

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Centromere identity: a challenge to be faced

The centromere is a genetic locus, required for faithful chromosome segregation, where spindle fibers attach to the chromosome through kinetochore. Loss of centromere or formation of multiple centromeres on a single chromosome leads to chromosome missegregation or chromosome breakage, respectively, which are detrimental for fitness and survival of a cell. Therefore, understanding the mechanism of centromere locus determination on the chromosome and perpetuation of such a locus in subsequent generation (known as centromere identity) is very fundamental to combat conditions like aneuploidy, spontaneous abortion, developmental defects, cell lethality and cancer. Recent studies have come up with different models to explain centromere identity. However, the exact mechanism still remains elusive. It has been observed that most eukaryotic centromeres are determined epigenetically rather than by a DNA sequence. The epigenetic marks that are instrumental in determining centromere identity are the histone H3 variant, CENP-A and the specialized posttranslational modification of the core histones. Here we will review the recent studies on the factors responsible for generating unique centromeric chromatin and how it perpetuates during cell division giving the present-day models. We will further focus on the probable mechanism of de novo centromere formation with an example of neocentromere. As a matter of similitude, this review will include marking extrachromosomal chromatin to be served as a partitioning locus by deposition of CENP-A homolog in budding yeast.     

Building the centromere: from foundation proteins to 3D organization

At each mitosis, accurate segregation of every chromosome is ensured by the assembly of a kinetochore at each centromeric locus. Six foundation kinetochore proteins that assemble hierarchically and co-dependently have been identified in vertebrates. CENP-A, Mis12, CENP-C, CENP-H and CENP-I localize to a core domain of centromeric chromatin. The sixth protein, CENP-B, although not essential in higher eukaryotes, has homologues in fission yeast that bind pericentric DNA and are essential for heterochromatin formation. Foundation kinetochore proteins have various roles and mutual interactions, and their associations with centromeric DNA and heterochromatin create structural domains that support the different functions of the centromere. Advances in molecular and microscopic techniques, coupled with rare centromere variants, have enabled us to gain fresh insights into the linear and 3D organization of centromeric chromatin.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes.

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

植物功能着丝粒DNA研究进展

着丝粒（centromere）是真核生物染色体的重要功能结构。在细胞有丝分裂和减数分裂过程中,着丝粒通过招募动粒蛋白行使功能,保障染色体正确分离和传递。真核生物中,含有着丝粒特异组蛋白的CenH3区域被定义为功能着丝粒区,即真正意义上的着丝粒。近年来,借助染色质免疫沉淀技术,人们对功能着丝粒DNA开展了深入研究,揭示其组成、结构及演化特征,并发现功能着丝粒区存在具有转录活性的基因,且部分基因具有重要生物学功能。由于存在大量重复DNA,着丝粒演化之谜一直未能完全揭示。对植物功能着丝粒DNA序列研究进展进行了概述,并重点阐述了着丝粒重复DNA研究的新方法和新进展,以期为深入开展相关研究提供借鉴。     

Replicating centromeric chromatin: spatial and temporal control of CENP-A assembly

The centromere is the fundamental unit for insuring chromosome inheritance. This complex region has a distinct type of chromatin in which histone H3 is replaced by a structurally different homologue identified in humans as CENP-A. In metazoans, specific DNA sequences are neither required nor sufficient for centromere identity. Rather, an epigenetic mark comprised of CENP-A containing chromatin is thought to be the major determinant of centromere identity. In this view, CENP-A deposition and chromatin assembly are fundamental processes for the maintenance of centromeric identity across mitotic and meiotic divisions. Several lines of evidence support CENP-A deposition in metazoans occurring at only one time in the cell cycle. Such cell cycle-dependent loading of CENP-A is found in divergent species from human to fission yeast, albeit with differences in the cell cycle point at which CENP-A is assembled. Cell cycle dependent CENP-A deposition requires multiple assembly factors for its deposition and maintenance. This review discusses the regulation of new CENP-A deposition and its relevance to centromere identity and inheritance.     

The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere

The centromere is essential for proper segregation and inheritance of genetic information. Centromeres are generally regulated to occur exactly once per chromosome; failure to do so leads to chromosome loss or damage and loss of linked genetic material. The mechanism for faithful regulation of centromere activity and number is unknown. The presence of ectopic centromeres (neocentromeres) has allowed us to probe the requirements and characteristics of centromere activation, maintenance, and structure. We utilized chromosome derivatives that placed a 290-kilobase "test segment" in three different contexts within the Drosophila melanogaster genome--immediately adjacent to (1) centromeric chromatin, (2) centric heterochromatin, or (3) euchromatin. Using irradiation mutagenesis, we freed this test segment from the source chromosome and genetically assayed whether the liberated "test fragment" exhibited centromere activity. We observed that this test fragment behaved differently with respect to centromere activity when liberated from different chromosomal contexts, despite an apparent sequence identity. Test segments juxtaposed to an active centromere produced fragments with neocentromere activity, whereas test segments far from centromeres did not. Once established, neocentromere activity was stable. The imposition of neocentromere activity on juxtaposed DNA supports the hypothesis that centromere activity and identity is capable of spreading and is regulated epigenetically.     

Centromeres of mammalian chromosomes

The centromere is the major cis-acting genetic locus involved in chromosome segregation in mitosis and meiosis. The mammalian centromere is characterized by large amounts of tandemly repeated satellite DNA and by a number of specific centromere proteins, at least one of which has been shown to interact directly with centromeric satellite DNA sequences. Although direct functional assays of chromosome segregation are still lacking, the data are most consistent with a structural and possibly functional role for satellite DNA in the mammalian centromere.     

Centromere dynamics and chromosome evolution in marsupials

The eukaryotic centromere poses an interesting evolutionary paradox: it is a chromatin entity indispensable to precise chromosome segregation in all eukaryotes, yet the DNA at the heart of the centromere is remarkably variable. Its important role of spindle attachment to the kinetochore during meiosis and mitosis notwithstanding, recent studies implicate the centromere as an active player in chromosome evolution and the divergence of species. This is exemplified by centromeric involvement in translocations, fusions, inversions, and centric shifts. Often species are defined karyotypically simply by the position of the centromere on certain chromosomes. Little is known about how the centromere, either as a functioning unit of chromatin or as a specific block of repetitive DNA sequences, acts in the creation of these types of chromosome rearrangements in an evolutionary context. Macropodine marsupials (kangaroos and wallabies) offer unique insights into current theories expositing centromere emergence during karyotypic diversification and speciation.     

Formation of a Functional Maize Centromere after Loss of Centromeric Sequences and Gain of Ectopic Sequences

The maize (Zea mays) B centromere is composed of B centromere–specific repeats (ZmBs), centromere-specific satellite repeats (CentC), and centromeric retrotransposons of maize (CRM). Here we describe a newly formed B centromere in maize, which has lost CentC sequences and has dramatically reduced CRM and ZmBs sequences, but still retains the molecular features of functional centromeres, such as CENH3, H2A phosphorylation at Thr-133, H3 phosphorylation at Ser-10, and Thr-3 immunostaining signals. This new centromere is stable and can be transmitted to offspring through meiosis. Anti-CENH3 chromatin immunoprecipitation sequencing revealed that a 723-kb region from the short arm of chromosome 9 (9S) was involved in the formation of the new centromere. The 723-kb region, which is gene poor and enriched for transposons, contains two abundant DNA motifs. Genes in the new centromere region are still transcribed. The original 723-kb region showed a higher DNA methylation level compared with native centromeres but was not significantly changed when it was involved in new centromere formation. Our results indicate that functional centromeres may be formed without the known centromere-specific sequences, yet the maintenance of a high DNA methylation level seems to be crucial for the proper function of a new centromere.     

Molecular hybridization to meiotic chromosomes in man reveals sequence arrangement on the no. 9 chromosome and provides clues to the nature of "parameres"

In situ hybridization of male human meiotic material has been used to elucidate the molecular organization of the centromeric region of human chromosome 9. The use of two cloned DNA sequences has shown that the centromere and the secondary constriction of this chromosome contain two separate repeated DNA families. The secondary constriction organizes into "paramere" bodies during pachytene. The individual parameres are comprised of one family of repeated DNA sequences.     

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae

At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.     

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae

At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.     

Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.     

Maize centromeres: where sequence meets epigenetics

The centromere is a highly organized structure mainly composed of repeat sequences, which make this region extremely difficult for sequencing and other analyses. It plays a conserved role in equal division of chromosomes into daughter cells in both mitosis and meiosis. However, centromere sequences show notable plasticity. In a dicentric chromosome, one of the centromeres can become inactivated with the underlying DNA unchanged. Furthermore, formerly inactive centromeres can regain activity under certain conditions. In addition, neocentromeres without centromeric repeats have been found in a wide spectrum of species. This evidence indicates that epigenetic mechanisms together with centromeric sequences are associated with centromere specification.