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Determining centromere identity: cyclical stories and forking paths
Author: Beth Sullivan
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"� 2001 Macmillan Magazines Ltd ?? I imagined a labyrinth of labyrinths, a maze of mazes, a twisting, turning, ever-widening labyrinth that contained both past and future?? 1 Chromosome inheritance should be understood because it affects the lives of all cells and organisms. Without proper chromosome segregation, functional germ cells and zygotes would not form, and somatic tissues and organs would not develop and differentiate. ANEUPLOIDY results from aberrant chromosome inheritance, and is an important component of human birth defects and an underlying cause of tumour progression 2,3 . Despite the key role of chromosome inheritance in cell and organ- ism viability and function, we know surprisingly little about the fundamental molecular mechanisms that mediate inheritance and how they go wrong in disease states. Nevertheless, this is an exciting time in chromo- some biology. Recently developed optical tools, fluores- cent labelling methods and real-time analyses, combined with studies of genetically tractable model organisms, have significantly advanced our understanding of chro- mosome structure and behaviour. However, more remains to be learned about this complex, mysterious and essential biological process. The CENTROMERE is central to the chromosome inheri- tance process. It was originally defined in 1880 by Walther Flemming as a cytologically visible ?primary? constriction in the chromosome 4 . In the early 1900s, centromeres were defined genetically as chromosomal sites that were essen- tial for normal inheritance and as regions of greatly reduced or absent meiotic recombination. We now know that the centromere is the site of kinetochore formation, the proteinaceous structure on each chromosome (FIG. 1) that is responsible for their attachment to and movement along microtubules; the centromere is therefore essential for chromosomal PLATEWARD PROMETAPHASE and POLEWARD ANAPHASE movements 5 . The terms centromere and kineto- chore are commonly used interchangeably, and many functions, DNA sequences and proteins have been called centromeric, even when there has been no evidence that they have a centromeric function. As optical methods and functional analyses have improved, it has become evident that the CENTROMERE REGION is structurally and functionally more complex than was previously thought. In this review, we use the term ?centromere? to refer specifically to the chromatin (DNA and proteins) that is responsible for kinetochore formation, and the term ?centromere region? in reference to the domains and functions present in the DETERMINING CENTROMERE IDENTITY: CYCLICAL STORIES AND FORKING PATHS Beth A. Sullivan*, Michael D. Blower* ? and Gary H. Karpen* The centromere is the genetic locus required for chromosome segregation. It is the site of spindle attachment to the chromosomes and is crucial for the transfer of genetic information between cell and organismal generations. Although the centromere was first recognized more than 120 years ago, little is known about what determines its site(s) of activity, and how it contributes to kinetochore formation and spindle attachment. Recent work in this field has supported the hypothesis that most eukaryotic centromeres are determined epigenetically rather than by primary DNA sequence. Here, we review recent studies that have elucidated the organization and functions of centromeric chromatin, and evaluate present-day models for how centromere identity and propagation are determined. *Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California 92037, USA. ? Department of Biology, University of California at San Diego, La Jolla, California 92037, USA. Correspondence to G.H.K. e-mail: karpen@salk.edu ANEUPLOIDY The presence of extra copies, or no copies, of some chromosomes. CENTROMERE The genetic locus required for chromosome segregation; contains DNA and proteins on which the kinetochore is formed. REVIEWS 584 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 585 REVIEWS spindle 6 . However, it is not clear how the centromere acts to form the kinetochore or how and why sites destined for centromere function are chosen (centromere identi- ty). What is the relative importance of primary DNA sequence compared with EPIGENETIC mechanisms? Which proteins are essential to centromere identity? How is cen- tromere identity replicated and what regulates the cycle of centromere propagation? This review describes recent advances in the study of molecular mechanisms of centromere identity. We pro- vide a brief overview of centromeric DNAs and present evidence that shows that they are neither necessary nor sufficient for kinetochore formation in multicellular eukaryotes. We then discuss recent studies that have described subdomains in the centromere region that mediate different inheritance functions. Finally, we eval- uate present-day models that account for the determi- nation and propagation of centromere identity. Outer kinetochore functions, chromosome movement, and sister-chromatid cohesion and separation are outside the focus of this review (see REFS 5,12 for recent reviews on these topics). Forking paths to centromere identity Many studies have focused on identifying ?the? sequences that confer centromere function (see REF. 13 and the references therein). The essential nature of cen- tromeres and their stable location in the chromosome had indicated that specific primary DNA sequences might determine centromere identity and propagation. These studies identified regions involved in centromere function by several approaches, for example by screen- ing for mutations that caused chromosome loss and that silenced centromeric marker genes, by functionally analysing chromosomal deletions and by de novo centromere assembly. The best-defined centromeric DNAs are in Saccharomyces cerevisiae. These simple, ?point? cen- tromeres ? 16 in total (one per chromosome) ? con- sist of an essential, conserved 125-bp sequence that comprises three functional elements that recruit cen- tromere proteins and organize them into cytologically invisible kinetochores 14 (FIG. 2a). Initially, it was expected that this dependence on pri- mary sequence for centromere function in S. cerevisiae would exist in other organisms; however, it is notably absent in other eukaryotes, although some functional and structural characteristics of centromeric domains are conserved. Generally, centromeres are surrounded by or embedded in heterochromatin, a repeat-rich, gene-poor region of the genome that normally represses the transcription of euchromatic genes (in a process called POSITION EFFECT VARIEGATION) 15 .In Schizosaccharomyces pombe, non-homologous 4?5-kb central core sequences are flanked by various inverted repeats that are shared between the three chromo- somes 16?18 (FIG. 2b). A minimum sequence of 25 kb, which contains the non-repetitive central core, inner repeats and a portion of the outer repeats, is an absolute requirement for centromere function and for stable chromosome transmission 19?21 . Deleting the inner vicinity of a centromere (centric HETEROCHROMATIN plus centromere, FIG. 1). The outer kinetochore (FIG. 1) encodes specific functions and is specifically responsible for microtubule interactions. It also mediates the transition from metaphase to anaphase, serving as the site of action for the SPINDLE ASSEMBLY CHECKPOINT (SAC) 6 . Defects in pro- teins involved in this checkpoint occur in human tumours, which indicates an important role for specific centromere functions in disease 7 . In addition, specialized sister-chromatid cohesion functions are associated with the centromeric region and are essential for proper reductional meiosis I divisions 8,9 . Centromeres/kinetochores come in several sizes and forms 10 (FIG. 2). Most eukaryotes have MONOCENTRIC chro- mosomes, the size of which can vary markedly between species (FIG. 2a?d). HOLOCENTRIC chromosomes (FIG. 2e) are present in organisms such as nematodes and crayfish 11 . Organisms such as centipedes have both holocentric and monocentric chromosomes in the same nuclei 10 . How and why such an essential cellular component has evolved into different structures and whether functional mechanisms are conserved between these different structures remains unclear (we discuss this issue further towards the end of this review). Generally, we understand the role that the kineto- chore has in mediating chromosome movement; it recruits the microtubule motor proteins, DYNEINS and KINESINS, which probably determine the direction and speed of chromosome movement along the micro- tubules 5 . We also know that key molecules involved in the SAC (such as Mad2 (mitotic arrest deficient 2) and Bub1 (budding uninhibited by benzimidazoles 1)) are mostly concentrated in the outer kinetochore, where they are appropriately positioned to signal the start of anaphase once all kinetochores are attached to the Centromere/inner kinetochore ? Centromere identity ? Centromeric chromatin ? Kinetochore structure Outer kinetochore ? Microtubule binding ? Prometaphase congression ? Anaphase movement ? Spindle attachment checkpoint Centric heterochromatin ? Centric cohesion Chromosome arms ? Anti-poleward forces ? Prometaphase congression ? Sister-chromatid cohesion Kinetochore microtubules Figure 1 | Structural and functional elements of the centromere region. The centromere/inner kinetochore, outer kinetochore, centric heterochromatin and chromosome arms, and associated functions, are shown. PLATEWARD METAPHASE MOVEMENTS (prometaphase congression). The movement of condensing chromosomes towards the metaphase plate, during which they capture spindle microtubules and orientate themselves in preparation for anaphase sister-chromatid separation. POLEWARD ANAPHASE MOVEMENTS The movement of chromatids along spindle microtubules towards the spindle poles. CENTROMERE REGION Chromatin and DNA in the vicinity of the functional centromere, including the pericentric heterochromatin. HETEROCHROMATIN A cytologically defined genomic component that contains repetitive DNA (highly repetitive satellite DNA, transposable elements and ribosomal DNA gene clusters) and some protein- coding genes. Most eukaryotic centromeres are embedded in heterochromatin. � 2001 Macmillan Magazines Ltd 586 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS below). Two studies have been instrumental in defining the boundaries 22 and functional subdomains 23 of cen- tromere 1 of S. pombe by assaying for the expression of marker genes that have been inserted across the centromere (discussed in more detail below). repeats compromises meiotic sister-chromatid segrega- tion 20 , showing that centromeric regions function in processes other than kinetochore assembly, and that kinetochore and cohesion domains are closely linked and important for proper chromosome segregation (see SPINDLE ASSEMBLY CHECKPOINT (SAC). A highly conserved surveillance mechanism in mitosis and meiosis that minimizes chromosome loss by preventing chromosomes from initiating anaphase until all kinetochores have successfully captured spindle microtubules. MONOCENTRIC When a kinetochore forms on a specific, limited region of a chromosome. HOLOCENTRIC When a kinetochore forms along the entire length of a chromosome. DYNEINS Microtubule-based molecular motors that move towards the minus end of microtubules. KINESINS Microtubule-based molecular motors that, in general, move towards the fast-growing, plus end of microtubules. EPIGENETIC Any heritable influence (in the progeny of cells or of individuals) on chromosome or gene function that is not accompanied by a change in DNA sequence. Examples of epigenetic events include mammalian X-chromosome inactivation, imprinting, centromere inactivation and position effect variegation. POSITION EFFECT VARIEGATION (PEV). The variable, heritable suppression of genes by their juxtaposition to heterochromatin or telomeres, or by movement of a gene into a different nuclear domain or chromosomal context. MINICHROMOSOME An extranumerary chromosome that contains functional elements, such as telomeres and centromeres, and is transmitted in meiosis and mitosis. a S. cerevisiae Cse4 125 bp d H. sapiens b S. pombe Cnp1 tRNA Outer OuterInner InnerCore Core tRNA 40?100 kb CENPA e C. elegans HCP-3 Telomere CENPA, CENPA homologues Transposons Direction of repeats Repetitive DNA Euchromatin Flanking heterochromatin Telomere 0.1?4 Mb c D. melanogaster 420 kb Figure 2 | Centromeric DNAs and conservation of CENPA at structurally distinct centromeres. The DNA sequence of centromeres differs between species, but the presence and function of CENPA and its homologues (shown in green) at kinetochores is highly conserved (c?d (right), chromosomes shown in blue). a | Saccharomyces cerevisiae centromere function depends on a region that contains three conserved elements (I, II, III), to which Cse4 localizes. b | Schizosaccharomyces pombe centromeres contain a unique central core, which Cnp1 localizes to, flanked by conserved inverted inner and outer repeats. c | The MINICHROMOSOME Dp1187, with the only defined Drosophila melanogaster centromere, consists of a core of 5-bp satellites and transposons, flanked by other repetitive DNA (red). Right, Cid (red) at endogenous fly centromeres and at minichromosomes (arrows). d | Human centromeres consist of alpha-satellite DNA (red arrows) tandemly arranged into higher- order repeats (blue arrows), which extend over megabases. CENPA localizes to a portion of these arrays. Right, a human cell line that contains a dicentric chromosome with one active (arrow) and one inactive (arrowhead) centromere. CENPB (red), a centromeric alpha-satellite-binding protein, is present at both centromeres. CENPA (green) locates to only endogenous centromeres and the active centromere of the dicentric chromosome. e | Caenorhabditis elegans kinetochores assemble along the length of each chromosome. Right, at metaphase, the centromeric histone HCP-3 (green) is present on the poleward-facing side of chromatids. CENPA, centromere-specific histone H3-like; Cid, Centromere identifier; Cnp1, S. pombe homologue of CENPA; Cse4, S. cerevisiae homologue of CENPA; and HCP-3, C. elegans homologue of CENPA. (Images courtesy of B. Sullivan (c,d), and L. Moore and M. Roth (e).) � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 587 REVIEWS mated the size or location of the centromere, because recombination suppression is a property of the flank- ing heterochromatin, rather than an intrinsic function or characteristic of the site of kinetochore forma- tion 37,38 . Nevertheless, A. thaliana studies have provided important descriptions of the structure and sequence composition of plant centromeric regions. Beet (genus Beta) centromeric regions have recently also been shown to consist of satellite DNA and transposable ele- ments 39 . Two beet minichromosomes that contain a subset of satellites found at normal centromeres show differential rates of meiotic transmission and should help to identify the minimal DNA elements required for Beta kinetochore assembly. Finally, in the nematode Caenorhabditis elegans,the holocentric chromosomes recruit and assemble cen- tromeric proteins along their lengths (FIG. 2e). Specific DNA sequences are seemingly not required as con- catamers of phage lambda DNA and many other types of DNA are stably transmitted 40 . Proteins are recruited into ?bundles? at prophase, then spread evenly on the poleward face of the chromosome arms by metaphase, which indicates that many areas of the C. elegans genome can support kinetochore assembly. Inactivated centromeres and neocentromeres The simple presence of centromeric DNA in a cell or on a chromosome does not automatically correlate with centromere function, as shown in humans and in flies. Dicentric chromosomes are engineered or natu- rally occurring abnormal chromosomes that contain two regions of centromeric DNA (for example, alpha- satellite DNA in humans) that are capable of function- ing as centromeres 41,42 . More than 60 years ago, Barbara McClintock described aberrant segregation of Drosophila centromeres are also located in repetitive DNA. So far, the only molecularly and functionally defined Drosophila centromere is on a 1.3-Mb X-derived minichromosome, Dp1187 (REFS 24,25) (FIG. 2c), progres- sive deletions of which have defined a 420-kb region that genetically confers normal chromosome inheritance. The primary repetitive DNA at human cen- tromeres is alpha-satellite DNA, which consists of a 171-bp monomer that is tandemly arranged into higher-order arrays that extend for 100 kb to several megabases 26 (FIG. 2d). The entire alpha-satellite array is unlikely to be involved in kinetochore assembly because antibodies to kinetochore proteins, such as CENPA (see TABLE 1 and below), localize to only a por- tion of the alpha-satellite DNA 27 . In addition, chro- mosomes that are naturally or artificially deleted for much of this array can still assemble a kinetochore and segregate normally 28?30 . Furthermore, not all alpha-satellite arrays form centromeres de novo (when in the form of human artificial chromosomes) 31,32 , indicating that other sequences or factors might be required to assemble and maintain functional human centromeres. However, de novo chromosome assembly seems to be most efficient when alpha-satellite DNA is introduced into human cell lines 31,32 , indicating that this repetitive DNA might be the best template for new centromere for- mation in cultured cells. Arabidopsis thaliana centromeres have only lately been localized to 500?1000-kb regions that contain 180-bp repeats, which are bounded by ribosomal DNA arrays and other repetitive sequences 33?36 . Genetic map- ping and recombination suppression have been used to localize these centromeres, rather than kinetochore function. These studies might therefore have overesti- Table 1 | Proteins involved in the functions of the centromere region* Location Function S. cerevisiae S. pombe C. elegans D. melanogaster Mammals Heterochromatin Heterochromatin Swi6 Su(var)2-5/Hp1 HP1 (humans), M31 (mouse) formation Chp1 Prod Histone H3 Clr4 Su(var)3-9 SUVAR39H1 (humans) methyltransferase Suvar39h1,2 (mouse) Sister-chromatid Mcd1, Scc1 Rad21, dRad21, Scc1 SCC1/hRAD21 cohesion Mis4,6,12 Mei-S332 Centromeric Centromere- Cse4 Cnp1 HCP-3 Cid CENPA (human) chromatin specific histone H3 Cenpa (mouse) CENPA loading Mis6 Kinetochore Inner plate Mif2 Cnp3 HCP-4 CENPC (human) Cenpc (mouse) Outer plate HCP-1,-2 Cmet CENPE (human),CENPF Cana Cenpe (mouse) Zw10 Rod *Proteins included in this table are only those referred to in this review. Known homologues (rows) are designated for various organisms (columns). For a more comprehensive list of proteins involved in chromosome inheritance, and in centromere and heterochromatin function, please see REF. 101. Full species names: Saccharomyces cerevisiae; Schizosaccharomyces pombe; Caenorhabditis elegans; Drosophila melanogaster. Cana, CenpE anaphase; CENPA, centromere protein A; CENPC, centromere protein C; CENPE, centromere protein E, kinesin-like; CENPF, centromere protein F; Chp1, chromodomain protein 1; Cid, Centromere identifier; Clr4, cryptic loci regulator 4; Cmet, CenpE metaphase; Cnp1,3, centromere proteins 1,3; HCP-1,-2,-3,-4, holocentric centromere proteins; Hp1, heterochromatin protein 1; M31, mammalian HP1; Mei-S332, meiotic-S332; Mif2, minichromosome fidelity 2; Mis4,6,12, mis-segregation 4,6,12; Prod, Proliferation disrupter; Rad21/Scc1/Mcd1, cohesins; Rod, Rough deal; Scc1/ SCC1, colon tumour susceptibility; Su(var)2-5,3-9/SUVAR39H1/Suvar39h1,2, suppressors of variegation (Note that the methyltransferase activity of Drosophila Su(var) 3-9 has not yet been shown); Swi6, switching gene 6; Zw10, zeste-white 10. CENPA A centromere-specific, histone H3-like protein. � 2001 Macmillan Magazines Ltd 588 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS spindle.) Human neocentromeres are found on natu- rally occurring, small, rearranged marker chromo- somes (see BOX 1), which are typically ascertained through prenatal screening or from cytogenetic analy- ses of individuals with developmental delay or congen- ital abnormalities 47 . These phenotypic abnormalities are probably caused by trisomy or tetrasomy for the genes that are encoded by the supernumerary chromo- somes, rather than by the presence of a neocentromere 47,50 . More than 40 human neocen- tromeres that involve 15 different chromosomes have been identified so far, indicating that many genomic regions are amenable to centromere activation or that any sequence can be activated as a centromere under certain conditions. A detailed study has shown that 22 centromere/kinetochore/centromere-region proteins are present at normal human centromeres and at neo- centromeres 51 , which indicates that neocentromeres might mediate inheritance through the same proteins and mechanisms as normal centromeres. dicentric chromosomes in maize, characterized by ANAPHASE BRIDGING, chromosome breakage and chromo- some loss 43 . However, dicentric chromosomes in humans (FIG. 2d) and in flies can be stably transmitted owing to the functional inactivation of one cen- tromere 27,41,44?46 or to centromere cooperation, when both centromeres remain functional and presumably act coordinately for kinetochore formation, micro- tubule attachment and anaphase segregation 41,42 . These studies explain the stable transmission of structurally dicentric chromosomes and show that the presence of centromeric DNA on a chromosome is not sufficient for centromere function. Centromeric DNAs are not always necessary for kinetochore formation, as shown by human and Drosophila chromosomes that lack typical centromeric DNA but can assemble kinetochores at sites called NEOCENTROMERES, and that are mitotically and meiotical- ly stable 47?50 .(Normally,ACENTRIC fragments are lost during cell division because they cannot attach to the ANAPHASE BRIDGES The physical stretching of dicentric chromosomes during anaphase due to the orientation and movement of linked kinetochores towards opposite spindle poles. NEOCENTROMERE Chromosomal sites that do not contain typical repetitive centromeric DNA but do acquire centromeric chromatin, can assemble kinetochores, can recruit other centromeric proteins, and are transmitted faithfully in meiosis and mitosis. ACENTRIC A chromosome or chromosomal fragment that lacks a centromere. Box 1 | Neocentromere formation in humans and in flies A naturally occurring human neocentromere derived from chromosome 10 is shown in part a of the figure. Human neocentromeres are often associated with gross chromosomal rearrangements; in this case, a large interstitial deletion that removed the middle part of chromosome 10. The order of events in this process is unclear; chromosomal deletions might occur first, followed by neocentromere formation (as shown), or neocentromere formation might occur on an intact chromosome, forming a dicentric that would then undergo rearrangements. Experiments have shown that the formation of experimentally induced neocentromeres in Drosophila melanogaster requires proximity to a functional centromere 57 . Part b of the figure shows the behaviour of a 320-kb euchromatic ?test fragment? in different contexts: the Dp1187 minichromosome derivative, Dp8-23; and its inversion derivative, ?238 (dashed lines/arrows show inversion breakpoints). Neocentromeres are only produced after the irradiation-induced breakage of ?238. They do not form when the test fragment is located far from the endogenous centromere before chromosome breakage, as in Dp8-23. Neocentromeres were also not recovered from the centric heterochromatin to the right of the centromere. These results indicate that neocentromere formation in flies might occur owing to the spreading of centromeric chromatin onto adjacent euchromatic DNA, but only when the flanking heterochromatin is eliminated. These studies also showed that once normally non- centromeric chromatin acquires neocentromere function, it is faithfully inherited through mitosis and meiosis. a Human chromosome 10 b D. melanogaster Alphoid DNA Kinetochore Neocentromere 0.4 Mb Irradiation No neocentromere formation Inversion Test fragment Euchromatin Centromere Dp8-23 Heterochromatin Marker genes ?238 Irradiation Irradiation No neocentromere formation Neocentromere formation � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 589 REVIEWS How are these normally non-centromeric regions activated? It is difficult to analyse the mechanism(s) of neocentromere formation in humans, as such cases are quite rare and presumably occur during meiosis in a parent. However, neocentromeres that bind centromere and kinetochore proteins can be experimentally induced on normally non-centromeric, euchromatic DNA after irradiation-induced breakage of a Drosophila minichromosome 24,49 (BOX 1). In such experiments, neo- centromere formation on a ?test fragment? that normal- ly lies more than 40 Mb from the endogenous X cen- tromere was assessed in different chromosomal contexts. The test fragment was only able to become a neocentromere if it was adjacent to the functional cen- tromere of the minichromosome before irradia- tion 49,56,57 . Therefore, neocentromere activation in Drosophila is thought to occur by the spreading in cis of centromeric proteins into adjacent, non-centromeric regions when heterochromatic BOUNDARY ELEMENTS (see Neocentromeres are often identified cytologically but only a few have been analysed molecularly. Recent studies 52?54 of human neocentromeric regions on three different chromosomes have defined the regions of CENPA binding to ~400 kb, remarkably similar to the size of the Drosophila minichromosome cen- tromere 52?54 . None of the activated neocentromeric regions differed in sequence composition from the parental homologous loci and the regions also did not acquire alpha-satellite DNA. Furthermore, these neo- centromeres did not show significant homology to each other, indicating that different sequences can acquire neocentromeric function. Interestingly, all three neo- centromeres were significantly (A+T)-rich (>60%) and enriched for retroviral elements, long terminal repeats and/or short tandem repeats. It is possible that (A+T)- rich DNA or repetitive arrays can more easily achieve the conformations required for centromere function and kinetochore assembly 55 . BOUNDARY ELEMENTS Chromatin that acts as an insulator to block changes in chromatin structure, protein binding, or the spreading of functional domains. Inner kinetochore HCP-3 (Cenpa) HCP-4 (Cenpc) Centromeric chromatin/ inner kinetochore Cid (Cenpa) Outer kinetochore Zw10 Polo kinase Cenp-meta & -ana (Cenpe) Rod Dynein Bub1 & Bub2 Sister chromatid cohesion Mei-S332 Rad21/Scc1 (cohesin) Flanking heterochromatin Hp1 Prod Su(Var)3-9 ? H3 Meth-Lys9 ? Flanking heterochromatin Swi6 (Hp1) Clr4 (Su(Var)3-9) H3 Meth-Lys9 Inner centromere Cnp1 (Cenpa) Mis6 Mis12 Kinetochore HCP-3 HCP-4 ? BUB-1, MCAK, HCP-1 Sister-chromatid cohesion Kinetochore Heterochromatin CidMei-S332 Hp1 Prod Polo, Rod, Bub1, Cenp-meta Heterochromatin Clr4, Rik1 Swi6, Chp1 Kinetochore Mis6 Cnp1 c S. pombea C. elegans b D. melanogaster Outer kinetochore HCP-1,-2 (Cenpf) BUB-1 MCAK Figure 3 | Structural and functional analyses of protein subdomains in the centromere region. The schematics show the location of different centromere- region proteins with respect to the DNA sequence, and the boxes summarize the epistatic relationships between them (arrows show localization dependency and red crosses indicate no dependency). Cnp1, Cid and HCP-3 are CENPA homologues; other homologies are indicated in parentheses. a | Prometaphase Caenorhabditis elegans holocentric chromosomes contain inner and outer kinetochore proteins distributed in clusters. Recruitment of outer kinetochore proteins is dependent on the presence of HCP-4, which requires HCP-3 for its localization, but not vice versa. b | Drosophila melanogaster centromere proteins. Cid is required for the recruitment of all outer kinetochore proteins and a sister-chromatid cohesion protein (Mei-S332), but not for that of proteins that associate with the flanking heterochromatin. Cid localizes to centromeres in the absence of these outer-kinetochore, flanking-heterochromatin and sister-cohesion proteins. c | Schizosaccharomyces pombe centromere proteins bind either the inner centromere (the centromere core and inner repeats), or the flanking heterochromatin. Mutation studies (see text) show these domains are functionally distinct. Mis6 is required to localize Cnp1; Clr4 and Rik1 are required to localize Swi6 and Chp1. BUB-1/Bub1,2, budding uninhibited by benzimidazoles 1; Chp1, chromodomain protein 1; Cid, Centromere identifier; Clr4, cryptic loci regulator 4; Cnp1, centromere protein 1; H3 Meth-Lys9, methylated lysine 9 of histone H3, mediated by Su(var)3-9 homologues; Hp1, heterochromatin protein 1; MCAK, mitotic centromere-associated kinesin; Mei-S332, meiotic-S332; Mis6,12, mis-segregation 6,12; Prod, Proliferation disrupter; Rod, Rough deal; Su(Var)3-9, suppressor of variegation 3-9; Swi6, switching gene 6; Zw10, zeste-white 10. � 2001 Macmillan Magazines Ltd 590 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS Several recent studies highlight the importance of CENPA proteins in the establishment and function of kinetochores in various organisms. Inactivating CENPA proteins in yeasts, worms, flies and mammals severely disrupts mitosis and cell-cycle progression 64,71,72 . Furthermore, inactivating or deleting CENPA causes the mislocalization of many kinetochore and centromere- region proteins: knocking it out in mice or inhibiting its homologue in C. elegans by RNA INTERFERENCE (RNAi) abolishes the ability of CENPC proteins to target the kine- tochore 72?74 . By comparison, disrupting CENPC in C. elegans has no effect on CENPA targeting (FIG. 3a).So, CENPA is upstream of CENPC in the kinetochore- assembly pathway in worms and in mice. In addition, the outer-kinetochore proteins in the worm ? BUB-1, MCAK and HCP-1 ? are mislocalized when CENPA is inhibited by RNAi 73,74 . Similarly, many Drosophila cen- tromere-region and outer-kinetochore proteins (such as Polo, Bub1, Cmet (CENP-meta), Rod (Rough deal) and MEI-S332 (Meiotic-S332)) are mislocalized in embryos and in cultured cells when the Drosophila homologue of CENPA (called Cid (Centromere identifier)) is inhibited by antibodies or by RNAi 71 (FIG. 3b). These data indicate that CENPA is a central component in kinetochore for- mation and centromere function. Understanding the biochemical mechanisms and proteins required to prop- agate CENPA-containing chromatin during or after DNA replication should improve our understanding of how centromere identity and function are maintained (see below). S. pombe centromere subdomains. Recent studies in S. pombe have indicated that the centromere is not a sin- gle locus, as previously thought, but has an unexpectedly fine structure (FIG. 3c). It is repressive for transcription at the inner and outer repeats and at the central core (FIG. 2b), but transcriptional silencing at the central core and at the outer repeats are mediated by different proteins 23 . Mutations in SWI6 and CHP1 (TABLE 1) alleviate the silencing of transgenes that have been inserted into the inner and outer repeats, but not of those inserted into the central core. Conversely, a mutation in MIS6 (TABLE 1) alleviates transgene silencing in the central core but not in the flanking repeats. Consistent with the genetic analysis of centromeric silencing, CHROMATIN IMMUNOPRECIPITATION (ChIP) analysis has shown that the centromere region is composed of non-overlapping protein domains: the inner and outer repeats contain Swi6, and Chp1, whereas the central core contains Mis6 (REF. 23) (FIG. 3c). The central core, which is the site of kinetochore formation and spin- dle attachment 75 , also contains Cnp1, the S. pombe homologue of CENPA (REF. 66). Proteins located in the outer repeats and in the central core are also essential for chromosome transmission, because mutations in mis6, mis12 (REF. 76), and swi6 (REF. 77) lead to increased chromo- somal loss and sister-chromatid non-disjunction. The inner-centromere and flanking-heterochromatin domains seem to be separated by boundary elements, perhaps encoded by tRNA genes (FIGS 2b, 3c). The S. pombe centromere regions are excellent models for further inves- tigation into the biochemical mechanisms responsible for below) are removed 57 . The mechanisms of human neo- centromere formation are less clear, but most human neocentromeres are reported to occur at sites that are significantly distant from the endogenous centromeres, as judged from metaphase chromosome analyses. Activation by the spreading of centromere proteins in cis might not be the mechanism for neocentromere forma- tion in humans. However, as the nucleus is dynamically compartmentalized 58?60 , perhaps neocentromere activa- tion in humans occurs in trans, through the inappropri- ate nuclear positioning of normally non-centromeric regions in centromeric nuclear domains 56,57 . Centromere plasticity is surprising; centromeric DNAs are not sufficient for centromere function and non-centromeric DNA occasionally acquires, then faith- fully propagates, centromere function. And yet cen- tromeres are essential elements that usually show a stable location and function. Neocentromere activation cannot be a frequent occurrence, otherwise monocentric chro- mosomes would become di- or multicentric and would be lost or become holocentric. Nevertheless, these obser- vations indicate that centromere identity might be estab- lished and propagated at a specific and consistent chro- mosomal site by epigenetic mechanisms 13,55 . The epigenetic determination and self-propagation of centromere identity can account for both centromere plasticity and stability. A centromeric epigenetic mark could be specified by exclusive protein binding, HISTONE modifications and/or by the spatial and temporal orga- nization of chromosomes or chromosomal processes, such as replication (as discussed in more detail below). However, the epigenetic determination of centromere identity and propagation, and the apparent lack of dependence on primary DNA sequence, do not rule out the possibility that sequence composition, such as enrichment for repeat sequences or an AT sequence bias, also has a role in these processes. Subdomains of centromeric chromatin Specification by CENPA.The designation of the site of centromere formation in higher eukaryotes might be determined through an epigenetic mechanism, but the specifics of this process remain unclear 13 . CENPA ? a component of the centromere/kinetochore (TABLE 1) ? was originally identified as an antigen that was recognized by human CREST ANTISERA; subsequent biochemical and molecular analyses showed it to be a histone-H3-related protein 61?63 . CENPA proteins are present in yeasts (S. cere- visiae 64,65 and S. pombe 66 ), C. elegans 67 and Drosophila 68 , representing an evolutionary link between the seemingly divergent centromeres of these organisms (FIG. 2). NUCLEO- SOMES can be assembled in vitro from purified CENPA and from histones H2A, H2B and H4 (REF. 69), consistent with previous observations, which indicates that CENPA nucleosomes are homotypic in vivo (that is, they contain two copies of CENPA, not one copy of H3 and one of CENPA) 70 . The homology of CENPA proteins to a core chromatin component (histone H3) and their presence at centromeres throughout the cell cycle make them a strong candidate for a protein that specifies and propagates the site of kinetochore assembly. HISTONE A family of small, highly conserved basic proteins, found in the chromatin of all eukaryotic cells, that associate with DNA to form a nucleosome. CREST ANTISERA Autoantibodies that recognize centromeric antigens (CENPs) found in the sera of patients with autoimmune diseases, such as CREST (calcinosis, Raynaud syndrome, oesophageal dysmotility, scleroderma and telangiectasia). NUCLEOSOME The fundamental unit into which DNA and histones are packaged in eukaryotic cells. RNA INTERFERENCE (RNAi). A process by which double-stranded RNA specifically silences the expression of homologous genes by degrading their cognate mRNA. CENPC A constitutive kinetochore protein. Its localization to the inner kinetochore is dependent on CENPA. MEI-S332 A centromere-region protein involved in sister-chromatid cohesion. SWI6 A chromodomain-containing Schizosaccharomyces pombe homologue of Drosophila heterochromatin protein 1. The chromodomain is a protein motif ? common to proteins that in some cases interact with chromatin ? that is involved in binding certain methylated histones; often associated with transcriptional repression. CHP1 A Schizosaccharomyces pombe chromodomain protein. MIS6 A centromere protein that binds to the central core of the Schizosaccharomyces pombe centromere and is required to establish or maintain centromeric chromatin structure. CHROMATIN IMMUNOPRECIPITATION (ChIP). A technique that isolates sequences from soluble DNA chromatin extracts (complexes of DNA and protein) using antibodies that recognize specific chromosomal proteins. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 591 REVIEWS require some property of heterochromatin. However, cytological studies in flies have shown that Cid-contain- ing chromatin is flanked by, but does not overlap with, chromatin that contains proteins involved in centric het- erochromatin function (Hp1) and centromere-region condensation (Prod (Proliferation disrupter)) (FIG. 3b and TABLE 1). Furthermore, mutations in Hp1 and prod do not affect Cid localization and vice versa 71 .So,Cid deposition at centromeres is independent of at least two key heterochromatin proteins. The presence of distinct centromere region domains is also supported by the observation that inhibiting the INNER CENTROMERE PROTEIN, Incenp, does not affect Cid deposition in Drosophila 83 . Similarly, in worms, INCENP is not dependent on HCP3 or HCP4 for its kinetochore localization and vice versa 74 (FIG. 3a).In Drosophila, Mei-S332 (REF. 8) (TABLE 1) is establishing and maintaining centromere region subdo- mains. In fact, recent studies have begun to elucidate the biochemical mechanisms involved in implementing and preserving the protein composition of the centromeric region 78?81 . For example, the selective methylation of his- tone H3 lysine 9 by SU(VAR)3-9 homologues (TABLE 1) has recently been found to create specialized binding sites for heterochromatin proteins, such as HP1 (heterochromatin protein 1, also called Su(Var)2-5), that are involved in marking chromatin states and organizing chromosomal domains 82 . Drosophila centromere subdomains. D. melanogaster centromeres are similarly organized. The positioning of centromeres in centric heterochromatin in most higher eukaryotes indicates that centromere function might HETEROCHROMATIN PROTEIN 1 (Hp1). A Drosophila heterochromatin protein that contains chromodomains. SU(VAR)3-9 A chromatin-binding translation initiation factor that suppresses position effect variegation in flies. Mammalian and Schizosaccharomyces pombe Su(Var)3-9 homologues are present in the centromere region of mitotic chromosomes, and encode a histone H3 methyltransferase. a c b Early S Nucleus Site of replication Centromere Centric heterochromatin H3 CENPA Mid S Late S Very late S Nucleus CENPA Centromere cluster H3 chromatin Mitosis Kinetochore assembly CENPA nucleosome H3 nucleosome CENPA loading factor H3 CAF CENPA H3 Replication Recruitment Nucleosome assembly Figure 4 | Models for the propagation of centromere identity. CENPA deposition determines centromere identity and propagation in these models, but other molecules or epigenetic marks could be responsible for these functions. a | The ?last to replicate? model. Centromeres are the only sequences replicated very late in S phase, when H3 is absent and CENPA is present. The opposite occurs in the ?earliest to replicate? model: CENPA is only present when centromeres replicate early in S (not shown). b | Nuclear-organization model. Centromeres are clustered and sequestered into one or more centromeric ?domains?, proposed to be the only site(s) of CENPA assembly in the nucleus. c | Cyclical chromatin assembly model. During replication, CENPA- and H3-containing nucleosomes segregate to daughter chromatids. Chromatin assembly factors (CAFs) or other loading factors then recruit CENPA and H3 to sites that already contain the appropriate histone to replenish nucleosome numbers. CENPA and H3 recruitment is unlikely to occur simultaneously as H3 assembly is coupled to replication, and CENPA assembly is not (see text). Nucleosome assembly and subsequent kinetochore assembly transmit centromeric chromatin through mitosis, where the replication and replenishment cycle begins again. � 2001 Macmillan Magazines Ltd 592 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS In S. cerevisiae, centromeric DNA has been reported to replicate in early S phase 90 . Cse4 (the S. cerevisiae homologue of CENPA) is expressed at low levels throughout the cell cycle 65 , but it is unclear at what point it is loaded onto chromatin and if centromere assembly is uncoupled from DNA replication. It is note- worthy that centromeres are not the earliest sequences to replicate in S. cerevisiae. In fact, they have been found to replicate approximately one-third of the way through the S phase, at a time when many non-centromeric DNAs are also replicated 90 .In S. pombe, Cnp1 expres- sion occurs from M to the G1/S boundary, before the peak of H3 synthesis at the onset of S phase 66 . Mis6 is required for proper localization of Cnp1 to centromeres (FIG. 3c) and acts during G1/S or at the G1/S boundary, therefore Cnp1 is probably incorporated into nucleo- somes before S phase commences. The precise replica- tion timing of centromeric DNA in S. pombe has not been reported; however, the implication from both yeast studies is that CENPA is incorporated into centromeric nucleosomes independently of DNA replication. Models that link centromere identity and propagation with very early or very late replication timing 88,89 have been directly tested using DNA-labelling methods based on the incorporation of thymidine analogues into repli- cating DNA. Studies of CENPA expression and the tim- ing of centromeric chromatin replication in cultured human cells 91 have shown that histone H3 expression peaks in early to mid-S phase, and CENPA mRNA and protein levels are highest in late S phase and G2. Inappropriate expression of CENPA in early S phase in human cells abolishes its specificity for centromeres, indi- cating that its synthesis and/or deposition might be regu- lated differently from bulk histones to allow for its exclu- sive incorporation into centromeric chromatin 70 . These results initially provided evidence in favour of the replica- tion-timing model for CENPA incorporation (FIG. 4a). However, human CENPA-associated chromatin has recently been shown to be neither the first nor the last region to be replicated; human centromeres replicate asynchronously in mid- to late S phase, simultaneously with non-centromeric DNA 91 . Furthermore, CENPA is actively assembled at centromeres in G2 (REFS 70,91).So, human centromere replication occurs broadly and asyn- chronously in S phase, before new CENPA is assembled into chromatin. As confirmation that centromeric chro- matin assembly is uncoupled from replication, new CENPA was shown to be assembled into centromeres even when DNA replication was blocked 91 . Similarly, a recent study in Drosophila showed that Cid is incorporat- ed into centromeres in the absence of DNA replication 89 . This study also indicated that centromeres in Drosophila cells cultured in vitro might replicate in early S phase and that H3 deposition is inhibited at this time. However, Drosophila centromeres have recently been found to replicate asynchronously in vivo, in mid- to late S phase, simultaneous with H3-containing chromatin 92 . If centromeric chromatin assembly in eukaryotes occurs independently of DNA replication timing, does physical sequestration of centromeres into unique nuclear domains allow exclusive incorporation of located near but not in the Cid-containing chromatin 71 (FIG. 3b), which provides a physical basis for a previous demonstration that kinetochore function and Mei-S332- mediated cohesion can be separated using minichromo- some derivatives 84 . Interestingly, Mei-S332 localization depends on the presence of functional Cid, but the absence of functional Mei-S332 has no effect on Cid cen- tromeric localization 71 (FIG. 3b). These analyses also showed that Cid has a key role in recruiting all outer- kinetochore proteins (see above) and a centric sister- chromatid cohesion protein. Likewise, in S. cerevisiae the localization of the cohesion protein, Scc1, also depends on an active kinetochore 85 . So, the centromere region consists of many, functionally distinct subdomains and additional studies are necessary to further elucidate their functional interactions. Centromere identity and DNA replication Do the apparent distinctions between yeast and multi- cellular eukaryotes, and between monocentric and holocentric chromosomes, reflect differences in the mechanisms that determine centromere identity? Possibly. However, we can begin to explain these differ- ences in a unified fashion by thinking about the propa- gation of centromere identity as a cyclical, epigenetic process. For instance, a region might be specified to form a centromere because it was packaged as cen- tromeric chromatin in the previous cell cycle 13,55 .To understand centromere identity and its propagation cycle, we first must understand how centromeric chro- matin, defined by the presence of CENPA, is replicated. Typically, new histones are deposited along DNA as it is synthesized during the S phase, owing to the activity of chromatin assembly factors (CAFs) 86,87 .Is centromere propagation dependent on the incorporation of CENPA into nucleosomes in place of H3? If so, then centromere propagation would be undermined by CENPA being incorporated into non-centromeric regions or by it being replaced by H3 at centromeres. Several models have indicated that centromere iden- tity might be propagated because CENPA-containing chromatin is temporally, spatially or physically separat- ed from sites of bulk DNA replication and/or histone deposition (FIG. 4a, b). In the ?last to replicate? model, CENPA is exclusively incorporated into centromeres as they are replicated in very late S phase, at a time when H3-containing chromatin is no longer replicating 70,88 (FIG. 4a). Another replication-timing model proposes that centromeres could be the first to replicate, before the replication of bulk H3-containing chromatin 89 . Nuclear-organization models have also been proposed, in which the specificity of CENPA incorporation into nucleosomes is accomplished by the physical sequestra- tion of centromere-assembly factors into isolated ?domains? inhabited by clusters of centromeres 89 (FIG. 4b), which prevent the inclusion of H3 into centromeric nucleosomes. Replication-timing and nuclear-organiza- tion models are not mutually exclusive, and another model has been proposed in which centromere seques- tration and replication timing combine to propagate centromere identity 89 . INNER CENTROMERE PROTEIN (Incenp). A family of proteins that transiently localize to the region between sister- chromatid kinetochores during mitosis. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 593 REVIEWS into centromeric nucleosomes is achieved through the activity of certain factors, is discussed below. The centromere identity cycle ?The only way I could surmise was that it be a cyclical, or circular, volume, a volume whose last page would be identical to the first, so that one might go on infinitely.? 1 Centromere identity studies started with ground-break- ing experiments on the small centromeres of S. cerevisiae. The seemingly simple dependence of this centromere on specific DNA sequences for its identity, and the conservation of molecules such as CENPA, led us to believe that the centromeres of higher eukaryotes would consist of larger numbers of S. cerevisiae-like cen- tromeres. We now know that higher eukaryotic centromeres are considerably larger than those in S. cerevisiae, and that the regulation of centromere iden- tity in organisms as distant as S. pombe, flies and humans is dependent on epigenetic marking and epigenetic propagation rather than on primary DNA sequence. The findings that human centromeres replicate asyn- chronously and that mammalian CENPA is synthesized in G2 have led to the proposal that the propagation of centromere identity might occur through the activity of a post-replication, CENPA-specific loading factor, per- haps equivalent to H3 CAFs. Such a centromere-identity loading factor might recognize CENPA-containing nucleosomes that were segregated to sister chromatids during replication and then deposit new CENPA onto each chromatid in the same region, replenishing the CENPA content of the chromatin 12 (FIG. 4c). Such a cycli- cal mechanism could account for the faithful propaga- tion of both monocentric and holocentric centromeres, and the fact that once CENPA and neocentromere iden- tity are acquired, they are propagated faithfully from one generation to the next 49,51,56 . The recruitment of flanking DNA into CENPA-containing chromatin can also account for the ability of centromeric chromatin to ?spread? or encroach onto non-centromeric DNA and form neocentromeres 56,57 . The key questions that remain to be answered are: whether a CAF or similar loading molecule exists that can deposit CENPA into centromeric nucleosomes; whether this function promotes the propagation of cen- tromere identity; and how these processes relate to the establishment and propagation of centromere region subdomains. The mislocalization of Cse4 in S. cerevisiae in ndc10 mutants 94 shows that the presence of CENPA at centromeres can be affected by other centromere pro- teins; however, controversy remains about the order in which S. cerevisiae kinetochore components are assem- bled 95?97 . S. pombe Mis6 (FIG. 3a and TABLE 1) is required for the proper localization of Cnp1 to the central core 66 , and thus might be directly or indirectly part of the epi- genetic mechanism that ensures the proper marking of the centromere. Identifying proteins that interact with CENPA should lead to a better understanding of the propagation of CENPA-containing chromatin and centromere identity. CENPA (FIG. 4b)? Studies in human and Drosophila interphase cells have shown that centromeres are not sequestered into specific replication domains or nuclear domains 91?93 . Data from yeast, Drosophila and human studies have also ruled out models of replication-depen- dent (FIG. 4a) or spatially dependent (FIG. 4b) deposition of CENPA and propagation of centromere identity. An alternative model, in which incorporation of CENPA a S. cerevisiae b S. pombe c D. melanogaster, human d C. elegans CENPA nucleosome H3 nucleosome HP1 SU(VAR)3-9 Kinetochore microtubule Figure 5 | A repeat-subunit and presentation model for centromeric chromatin in different species. a | The Saccharomyces cerevisiae centromere probably consists of a single, Cse4-containing nucleosome that gives rise to the kinetochore. b | The Schizosaccharomyces pombe centromere consists of multiple Cnp1-containing nucleosomes that are flanked by heterochromatin. The inverted-repeat structure of the centromere-flanking regions indicate that the centromere region might form a stem?loop structure 97 , perhaps stabilized by heterochromatic proteins, such as homologues of HP1 and SU(VAR)3-9. c | The larger and more complex centromeres of Drosophila melanogaster and humans possibly contain repeats of similar ?loops?, or related higher-order structures, that are concentrated at specific chromosomal regions. Because tandem, rather than inverted, repeats exist in fly and human centromeric DNA, they might not form stem?loop structures; the DNA might, for example, spiral through a centromeric higher-order structure. Flanking heterochromatin might form a boundary that limits the spreading of centromeric chromatin in monocentric chromosomes 57 (see BOX 1). Such boundaries might distinguish monocentric from holocentric centromeres. d | Caenorhabditis elegans holocentric chromosomes might contain more repeat units at multiple sites, covering most of the chromosome. In these models, the higher-order structure of centromeric chromatin is conserved, and might be required to ?present? the centromeric chromatin to the outer face of condensed chromosomes (see text for more details). CENPA, centromere-specific, histone H3-like protein; Cnp1, S. pombe homologue of CENPA; Cse4, S. cerevisiae homologue of CENPA; HP1, heterochromatin protein 1; SU(VAR)3-9, suppressor of variegation 3-9. � 2001 Macmillan Magazines Ltd 594 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS chromosomes evolved from monocentric chromo- somes, owing to the loss of heterochromatic bound- ary elements and by the in cis spreading of centromer- ic chromatin, in a manner analogous to the generation of Drosophila neocentromeres 57 (BOX 1). Why are S. cerevisiae centromeres different from other well-studied centromeres, despite the presence of the CENPA orthologue Cse4? The apparent absence of epigenetic effects at S. cerevisiae centromeres might relate to the fundamental composition of the cen- tromeric chromatin; unlike higher eukaryotes, it is believed that the S. cerevisiae centromere contains a single, Cse4-containing nucleosome 64 (FIG. 5a). Replication of a single-nucleosome centromere would produce daughter strands, of which only one sister chromatid is likely to contain a Cse4-containing nucle- osome. A cyclical mechanism that requires the recog- nition of existing centromeric chromatin to deposit new CENPA nucleosomes (FIG. 4c) would not work if one chromatid completely lacked CENPA nucleo- somes. So, a primary-sequence-dependent mechanism might be necessary to ensure that both products of replication can recruit Cse4 and other proteins. Nevertheless, it is important to note that no direct tests for epigenetic mechanisms have been reported for S. cerevisiae centromeres, so it is still possible that these simple centromeres are also subject to some kind of epigenetic regulation. Conclusion This is an exciting time for centromere biologists. The tools are now assembled to do in-depth analyses of the biochemical mechanisms that determine centromere identity and kinetochore formation. Studies aimed at identifying CENPA-interacting proteins, their bio- chemical activities, and the role of centromeric DNA are essential. In the next few years, we should have a significantly greater understanding of these funda- mental biological processes, bringing this cycle of sto- ries and forking paths to a more satisfying conclusion. Understanding the basic biology of centromere struc- ture and function is also of relevance to human health: by identifying key molecules involved in birth defects and cancer 2,7 , this research could lead to new diagnos- tic tools and treatments. Furthermore, a more com- plete understanding of centromeres and chromosome inheritance in multicellular eukaryotes will lead to the efficient, controlled use of artificial chromosome cloning vectors 26,31,32 ; the ability to transfer large domains and genes into animal and plant cells as intact, functional chromosomes would be extremely useful for engineering livestock and crops, and for developing gene therapy vectors for use in humans. The inverted-repeat structure of the heterochromatin that flanks S. pombe centromeres and the behaviour of deletion constructs led to the proposal that the S. pombe centromere region might assume a stem?loop structure in which the core is in the loop, and the outer and inner repeats are in the stem 98 (FIG. 5b). The S. pombe cen- tromere provides an attractive model for centromere organization on a small scale, but how can this model be extrapolated to the centromeres of higher eukaryotes that are much larger and how can this organization be reconciled with the holocentric chromosomes observed in some organisms? The repeat-subunit model of cen- tromere organization proposed by Zinkowski et al. 99 provides an explanation for the evolutionary conserva- tion of centromere proteins between the different cen- tromere/kinetochore structures seen in many organisms. This model was originally proposed to explain the fact that CREST antibody staining appeared punctate and discontinuous in stretched chromosomes, whereas the underlying alpha satellite array was uniform, indicating that the centromere might not be a single entity but a series of basic, repeated subunits. The repetitive and interspersed form of the cen- tromere is especially obvious in the holocentric chro- mosomes of C. elegans (FIG. 5d) where HCP-3 (the C. elegans homologue of CENPA) is present in a few discrete foci in interphase cells, which then coalesce to cover the poleward face of the chromosomes during mitosis, forming a linear kinetochore ribbon 67 (FIG. 2e). Monocentric centromeres might be organized in a similar fashion (FIG. 5b, c), but the size and number of repeats might be limited by the presence of hete- rochromatic boundary elements. At S. pombe and Drosophila centromeres, each Cenpa-containing chro- matin domain is flanked by heterochromatin that contains proteins such as Hp1 and Su(var)3-9 (FIG. 3). Because neocentromere formation in Drosophila requires proximity to a functional centromere and the absence of intervening heterochromatin (BOX 1), it is possible that the heterochromatin itself provides a boundary between centromeric and adjacent chro- matin 57 . In addition, the heterochromatin could be responsible for looping the centromeric DNA 100 into a higher-order structure in such a way that the CENPA- containing chromatin is located on the poleward face of the chromosome and beneath the outer kineto- chore proteins, ?presenting? them to the nucleoplasm to mediate microtubule interactions (FIG. 5c). This model can account for the differences in the localiza- tion of proteins and their different gene-silencing effects, the requirement for both heterochromatin and CENPA-containing chromatin for complete cen- tromere function, the generation of neocentromeres and the evolution of holocentric chromosomes. Holocentric chromosomes could represent the first centromeres; kinetochore formation probably evolved first with random sequence specificity, followed by the evolution of monocentric chromosomes that arose owing to transposon invasion, to satellite DNA expan- sion and to the formation of flanking heterochro- matin. However, it is also possible that holocentric Links DATABASE LINKS Mad2 | Bub1 | CENPA | BUB-1 | MCAK | HCP-1 | Polo | Cmet | Rod | Mei-S332 | Cid | Swi6 | Chp1 | Mis6 | Cnp1 | mis12 | Hp1 | Prod | INCENP | Scc1 | Cse4 | ndc10 | HCP-3 | Clr4 | Rik1 FURTHER INFORMATION Walther Flemming | Barbara McClintock | Centromere research labs | Gary Karpen?s lab � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 595 REVIEWS 1. Borges, J. L. in Jorge Luis Borges: Collected Fictions 125 (Penguin Books, New York City, 1998). An example of metafiction dealing with cycles of life, written by a master of the genre. 2. Hassold, T. & Hunt, P. To err (meiotically) is human: the genesis of human aneuploidy. Nature Rev. Genet. 2, 280?291 (2001). 3. Mitelman, F. Catalog of Chromosome Aberrations in Cancer (Wiley, New York, 1994). 4. Flemming, W. Beitrag zur Kenntnis der Zelle und ihrer Lebenserscheinungen, Teil II. Archiv. Mikrosk. Anat. 18, 151?259 (1880). 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A solid foundation: functional specialization of centromeric chromatin. Curr. Opin. Genet. Dev. 11, 182?188 (2001). 13. Karpen, G. H. & Allshire, R. C. The case for epigenetic effects on centromere identity and function. Trends Genet. 13, 489?496 (1997). 14. Hyman, A. A. & Sorger, P. K. Structure and function of kinetochores in budding yeast. Annu. Rev. Cell Dev. Biol. 11, 471?495 (1995). 15. Weiler, K. S. & Wakimoto, B. T. Heterochromatin and gene expression in Drosophila. Annu. Rev. Genet. 29, 577?605 (1995). 16. Clarke, L., Amstutz, H., Fishel, B. & Carbon, J. Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe. Proc. Natl Acad. Sci. USA 83, 8253?8257 (1986). 17. Nakaseko, Y., Adachi, Y., Funahashi, S., Niwa, O. & Yanagida, M. Chromosome walking shows a highly repetitive sequence present in all the centromere regions of fission yeast. EMBO J. 5, 1011?1021 (1986). 18. Nakaseko, Y., Kinoshita, N. & Yanagida, M. 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Cell 79, 865?874 (1994). The first clear demonstration of epigenetic effects on the function of the Schizosaccharomyces pombe centromere. 99. Zinkowski, R. P., Meyne, J. & Brinkley, B. R. The centromere?kinetochore complex: a repeat subunit model. J. Cell Biol. 113, 1091?1110 (1991). A classic paper on centromere structure that proposes that the centromere/kinetochore is composed of repeating subunits. 100. Seum, C., Delattre, M., Spierer, A. & Spierer, P. Ectopic HP1 promotes chromosome loops and variegated silencing in Drosophila. EMBO J. 20, 812?818 (2001). 101. Dobie, K., Hari, K., Maggert, K. & Karpen, G. H. Centromere proteins and chromosome inheritance: a complex affair. Curr. Opin. Genet. Dev. 9, 206?217 (1999). Acknowledgements We thank K. Sullivan for critical comments on the manuscript, and L. Moore and M. Roth (Fred Hutchison Cancer Research Center, Seattle) for the image in figure 2e. Our research on centromeres is supported by grants from the National Institutes of Health. "
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