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Doi:10.1016/j.febslet.2008.04.014

FEBS Letters 582 (2008) 1950–1959 Centromeres: Old tales and new tools P. Vagnarelli*, S.A. Ribeiro, W.C. Earnshaw* Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, KingÕs Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Received 3 April 2008; accepted 11 April 2008 Available online 22 April 2008 Edited by Ulrike Kutay point centromeres bind a single microtubule. (b) In regional The centromere is a specialised region of the eukary- centromeres (Shizosaccharomyces pombe, Drosophila melano- otic chromosome that directs the equal segregation of sister chro-matids into two daughter cells during mitosis. In mitosis, the gaster, Gallus gallus, Homo sapiens), the centromeric region kinetochores mediate (1) microtubule capture and chromosome is composed of much longer DNA arrays, and forms a larger alignment at a metaphase plate; (2) the correction of improper kinetochore that interacts with a number of microtubules. Re- microtubule attachments; (3) the maintenance of an active check- gional centromeres evolve naturally in association with charac- point until bi-orientation is achieved by the whole complement of teristic underlying DNA sequences, but are not absolutely chromosomes; (4) the establishment of tension within the centro- restricted to particular DNA sequences. In the diffuse centro- mere which, in turn, contributes to silencing of the spindle check- mere of holocentric chromosomes (e.g. Caenorhabditis ele- point and triggers the onset of anaphase.
gans), microtubule-binding kinetochore determinants are In this review, we will analyse how centromeres are organised distributed along the length of the chromosome with respect to chromatin types and arrangements.
Ó In this review, we will analyse how centromeres are organ- 2008 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved.
ised with respect to chromatin types and arrangements. Forother analyses of how the kinetochore is formed and organised Keywords: Centromere; Kinetochore; CENP-A; Histone we refer the reader to other reviews .
2. In the beginning there was DNA. .
Despite the high conservation of the segregation machinery among eukaryotes, specific centromere-associated DNAs arehighly divergent and evolve rapidly during speciation. In S.
The centromere is a specialised region of the eukaryotic cerevisiae, the centromeric DNA (CEN sequence) comprises chromosome that directs the equal segregation of sister chro- 125 bp and consists of three elements: CDEI, CDEII and matids into two daughter cells during mitosis. The centromere CDEIII . In S. pombe, the centromere is composed of a appears as a cytologically visible primary constriction on mito- central core element (ctr) of 4–7 kb, and is flanked by imr se- tic chromosomes of higher eukaryotes. The outer surface of quences and otr outer repeats with an overall size range of 30– the centromere is the site where the kinetochore assembles: this 110 kb, depending on the chromosome . In higher represents the major machinery responsible for chromosome eukaryotes, the centromeric DNA is normally characterised segregation and is where interactions with microtubule plus by highly repetitive tandem sequences. Because of this repeti- ends occur. In mitosis, the kinetochores mediate (1) microtu- tive organisation, the full sequence of a natural vertebrate cen- bule capture and chromosome alignment at a metaphase plate; tromere has yet to be achieved Nevertheless, several (2) the correction of improper microtubule attachments; (3) the studies have contributed to understanding the general rules maintenance of an active checkpoint until bi-orientation is for vertebrate centromeric DNA composition and organisa- achieved by the whole complement of chromosomes; (4) the establishment of tension within the centromere which, in turn, In general, human centromeric DNA consists of arrays com- contributes to silencing of the spindle checkpoint and triggers prising 0.2–7 megabases of a 171 bp a-satellite motif repeated the onset of anaphase in tandem head-to-tail arrangement . This a-satellite There are two different types of centromeres in eukaryotes: DNA is present at all natural human centromeres .
localised centromeres and diffuse centromeres () a-Satellite can be subdivided in two types . Type I forms The former can be subdivided into two variants. (a) In point regular higher order repeat arrays (). Type II is mono- centromeres (Saccharomyces cerevisiae), kinetochore for- meric a-satellite and lacks a regular higher-order organisation.
mation on a discrete stretch of centromeric DNA is rigorously Type I a-satellite DNA is associated with centromere function specified by the underlying DNA sequence. Kinetochores of and contains a 17 bp motif known as the CENP-B box whichrepresents the binding site for the centromeric protein CENP-B Type II a-satellite DNA usually flanks the type I *Corresponding authors. Fax: +44 (0) 131 650 7100.
array and is often interrupted by LINE and SINE sequences E-mail addresses: (P. Vagnarelli), ed.ac.uk (W.C. Earnshaw).
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2008.04.014

P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 Fig. 1. Centromere organisation in eukaryotes. The DNA sequence of centromeric regions differs between species but the presence of CENP-A andits homologues (shown in orange) at the kinetochores is conserved. (A) S. cerevisiae has a point centromere that encompasses 125 bp and is composedof three conserved elements (CDEI, CDEII, CDEIII). This is wrapped around a single nucleosome containing the CENP-A homolog Cse4. Eachkinetochore in budding yeast makes only one stable microtubule attachment during metaphase. (A0) Meiotic chromosome spread of S. cerevisiae.
Red - Ndc10 kinetochore protein; Blue - DNA. Picture kindly provided by A. Marston, University of Edinburgh. (B) S. pombe has a regionalcentromere. The kinetochore assembles over the central domain, which contains the non-repetitive core (violet box) where Cnp1 localizes,surrounded by the innermost repeats (IMR; yellow box arrows). The outer repeats (OTR; blue box arrows) flank the central domain and are boundin heterochromatin. Each kinetochore interacts with 2–4 microtubules. (B0) Chromosomes of S. pombe. Red – Cnp1; Blue – DNA. Image kindlyprovided by A. Pidoux, University of Edinburgh. (C) H. sapiens has a more complex regional centromere. Two families of a-satellite DNA (red andgrey arrow boxes) are found only in the centromeric region. Type-I a-satellite (red box arrow) is composed of regular chromosome-specific higher-order repeating units, is characterized by the presence of CENP-B boxes (white triangles), and associates with CENP-A. Type-II a-satellite (grey boxarrows) consists of diverged alphoid monomer units (different coloured triangles). Between 20 and 30 microtubules attach to each humankinetochore. (C0) Partial metaphase spread of human chromosomes. Red – CENP-A; Blue – DNA. (D) C. elegans chromosomes have diffusecentromeres. CENP-A colocalizes with microtubule binding sites along the length of each chromosome. (D0) Metaphase chromosomes of C. elegans.
Red – Knl1; Blue – DNA. Image kindly provided by R. Gassmann, University of San Diego.
If the DNA sequence itself has not thus far given clues to the This was first revealed by the analysis of stable dicentric foundation for either the establishment or the maintenance of chromosomes . These chromosomes are the products a functional centromere, the analysis of ‘‘abnormal centro- of duplications or more complex chromosome re-arrangements meres'' identified by classical cytogenetics has opened the field and contain two regions of sequence normally found within to the concept that epigenetic factors are involved in the cen- functional centromeres (they can be either from the same or tromeric specification and propagation.
different chromosomes). In the case of dicentric chromosomes

P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 containing two autosomal centromeres, both centromeric Taken together these observations reveal that neither specific DNA arrays bind the protein CENP-B, however one of the DNA sequences (e.g. a-satellite) nor the DNA binding protein two centromeres is subject to inactivation (centromere inacti- CENP-B are essential or sufficient to dictate the assembly of a vation) and therefore does not bind markers for the kineto- chores, such as CENP-C and is no longer capable to However, studies of de novo chromosome formation follow- interact with the spindle microtubules. In the case of dicentric ing the introduction of long stretches of cloned DNA into cells, X chromosomes, the inactive centromere may fail to bind even have shown that even if particular DNA sequences are not the CENP-B protein. This led to the initial proposal that cen- absolutely required, strong preferences govern experimental tromere inactivation in these chromosomes might be caused by centromere formation on naked DNA For example, de modifications in chromatin structure (e.g. be epigenetic) novo centromere assembly only occurs on type I a-satellite The acquired chromatin modifications that lead to centromere DNA . These studies also revealed that the CENP-B inactivation are inherited from one cell division to the next, box plays an essential role in the establishment but not in therefore rendering this type of dicentric chromosome stable.
the maintenance of a functional centromere (see below) Further evidence for epigenetic effects in centromere specifi- We therefore conclude that in contrast to yeast point centro- cation came from the analysis of neo-centromeric chromo- meres, where DNA sequences specify sites of centromere somes . These chromosomes are the products of assembly, in regional centromeres some DNA characteristics, complex re-arrangements in which an interstitial euchromatic yet to be fully understood, specify a chromatin conformation, chromosome region acquires the function of centromeric which via epigenetic mechanisms controls the formation and DNA and nucleates the formation of a kinetochore. An early the maintenance of a functional centromere. Indeed, such epi- and unpublished analysis of one such neocentromere is shown genetic mechanisms can also operate on the regional centro- in (see also These chromosomes lack a character- mere of the fission yeast . Future studies aimed at istic centromeric primary constriction and may, as in the case elucidating the mechanisms of de novo chromosome formation shown, have smaller kinetochores than the normal chromo- and maintenance in vertebrates coupled with more extensive somes. Because euchromatic chromosome arm sequences lack sequence analyses of different neo-centromeric chromosomes CENP-B boxes (as is the case for the a-satellite array found on and evolutionary studies of centromere ‘‘drift'' during specia- the Y chromosome both neocentromeres and the Y cen- tion may eventually identify patterns of DNA sequence re- tromere fail to bind CENP-B (). Once formed, neocen- tromeres are fully functional and stably maintained through approaches will be required to map out the epigenetic ‘‘land- mitosis and meiosis .
scape'' that specifies centromere activity.
Fig. 2. A human chromosome in which a neocentromere has formed in euchromatin of a marker chromosome derived from chromosome 11. (A, A0)The marker chromosome (m) has a centromere that is labelled similar to that of the Y chromosome using anti-centromere autoantibodies that reactwith CENP-A, CENP-B and CENP-C. (B, B0) The centromere of both the marker and Y chromosomes fails to react with an antibody specific forCENP-B. This figure comes from a study performed in 1989, but not published until much later .
P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 3. A special chromatin flavour for centromeric chromatin assembly . In vivo studieshave shown that nucleosomes containing CENP-A generally Despite the fact that common themes have yet to be identi- lack histone H3. Since Cse4 interacts genetically with H2A fied in the primary sequence of centromeric DNAs, important and H4 and co-purifies with H2A, H2B and H4 it was gener- similarities in chromatin organisation are shared by all eukary- ally assumed that the centromeric-specific nucleosome is com- otic centromeres. All centromeres contain a centromere-spe- posed of two molecules each of Cse4, H2A, H2B and H4.
cific histone H3 variant, CENP-A (reviewed in However, a recent study has reported that nucleosomes con- This protein has subsequently been given other names, includ- taining Cse4 possess an unusual composition, with H2A and ing Cse4 (S. cerevisiae) , CID (D. melanogaster) H2B replaced by one molecule of Scm3 , a protein first ceHcp-3 (C. elegans) and HTR12 (Arabadopsis thali- identified as dosage suppressor of a temperature-sensitive Cse4 allele in Saccharomyces cerevisiae. Smc3 can also displace CENP-A is found not only at all natural centromeres in ver- H2A and H2B from pre-assembled canonical octamers con- tebrates but also at the variant functional centromeres we have taining Cse4 in vitro. These results suggest that the budding described above (e.g. neo-centromeres and de novo artificial yeast centromeric nucleosome may be a hexamer containing centromeres) . Studies of CENP-A knock-outs in mouse two copies of Cse4, Scm3 and Histone H4. These exciting DT40 C. elegans and by RNAi in human cells new findings could represent a specialised situation that occurs have clearly shown that CENP-A is essential for the estab- only in the single nucleosome of the budding yeast point cen- lishment and maintenance of centromeric function. All these tromere. To date, orthologs of Scm3 have not been found in studies therefore place CENP-A at or near the top of the hier- other eukaryotes apart from fungi. However, these recent ad- archical pathway that directs centromere/kinetochore forma- vances in the understanding of budding yeast centromere tion and activity.
structure suggest the possibility that the CENP-A-containing CENP-A chromatin is localized in the inner kinetochore chromatin could also be quite ‘‘special'' in higher eukaryotes.
plate (Carol A. Cooke and WCE, unpublished data), a part Still on the quest for the special flavour of CENP-A contain- of the electron-dense kinetochore structure visible at the pri- ing chromatin, a recent study on Drosophila centromeric chro- mary constriction of eukaryotic chromosomes. The outer kine- matin organisation has produced provocative results. Based on tochore does not contain detectable DNA In vitro studies data obtained from cross-linking experiments and atomic force suggest that nucleosomes containing CENP-A adopt a struc- microscopy it was proposed that the nucleosomes containing ture that exhibits greater conformational rigidity than conven- Cid/CENP-A prepared from interphase cells are composed tional chromatin, as judged by its deuterium exchange rate of heterotypic tetramers termed ‘‘hemisomes'', and not canon- relative to nucleosomes containing H3 How these in vitro ical octamers These experiments are controversial, as the findings relate to the in vivo organisation and behaviour of the results could be influenced by the distribution of cross-linkable CENP-A chromatin is still not clear. This special centromeric residues in the Cid/CENP-A nucleosome However, an chromatin may have a more compact structure which func- octameric histone arrangement was detectable as a minor tions as a platform for kinetochore assembly . This plat- cross-linked species when CENP-A-containing nucleosomes form must be structurally distinct and physically robust in were prepared from mitotic cells.
order to maintain its architecture when tension within the spin- In other experimental systems and using a range of analyses, dle pulls the sister kinetochores towards opposite spindle poles most results obtained to date are consistent with CENP-A during metaphase.
occurring in canonical nucleosomes. In one study, CENP-A In regional centromeres, 100–1000 kilobases of DNA (100– chromatin was obtained from interphase nuclei by immuno- 300 kb estimated from ChIP studies on neocentromeres precipitation using a monoclonal antibody In another and 1 Mb from studies using fibre-FISH on artificial study, CENP-A-containing nucleosomes were isolated by chromosomes are organised in CENP-A containing chro- means of a tag linked to an over-expressed form of CENP- matin. These size discrepancies could either reflect the intrinsic A. Therefore, the endogenous protein was still present and technical limitations of the differing approaches used or the data are generally lacking concerning the functionality of the different substrates examined. Despite the differences, a com- engineered protein apart from its localisation In some mon theme seems to emerge – CENP-A nucleosomes are not cases, only the tagged version of CENP-A was recovered in arranged along a linear uninterrupted stretch of chromatin. In- pulldowns together with H2A, H2B and H4. However, H3 stead, they appear to be clustered, with each cluster spaced was also present in the purified samples although it was less from the next by a region of CENP-A-free chromatin abundant. From these experiments it was concluded that in In at least some cases it appears that nucleosomes human cells CENP-A nucleosomes are typically found in containing H3K4Me2 (histone-H3 di-methylated on Lys 4) homotypic octamers with only about 10% found in heterotypic occupy the CENP-A-negative gaps within the CENP-A chro- matin region This organisation for the centromeric chro- Once CENP-A is incorporated into centromeric chromatin it matin was unexpected since H3K4Me2 is a modification remains stable across the whole cell cycle including during characteristic of euchromatic regions In the future, DNA replication. It has recently been demonstrated that load- more extensive studies using different approaches will be re- ing of new CENP-A into the replicated centromeres happens quired to develop a general model for the organisation of the only in late mitosis or early G1 and not in S or CENP-A chromatin block at vertebrate centromeres.
G2 as originally proposed . Therefore, new incorporation In budding yeast (the system in which centromeric chroma- of CENP-A occurs only after cells pass through mitosis. The tin organisation has been analysed in the greatest detail), a nature of the priming event that happens during mitosis and single nucleosome containing Cse4 forms the minimal unit is required for new CENP-A assembly is still unknown – pro- P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 Fig. 3. Chromatin subdomains within the centromeres are characterised by different histone modifications and have different functions. (A, B)Chicken metaphase chromosomes stained for CENP-A (green) and H3K9Me3 (red) (A) or H3T3ph (red) (B). These histone modifications are presentonly in the inner centromeric chromatin of mitotic chromosomes. (C) Human metaphase chromosome stained for CENP-A (green) and HP1-a (red).
HP1-a is enriched in the centromeric and peri-centromeric heterochromatin. (D) Human mitotic chromosome stained for CENP-A (green) andH3K4Me2 (red). This histone modification is typical of euchromatin regions, however in rare instances it also appears to co-localise with CENP-Asignal (arrows) and inset. Picture kindly provided by S. Cardinale, University of Edinburgh. (E) Schematic representation of the primary constrictionof a regional centromere where the localisation of different histone modifications is indicated (left). Known functions, which are attributed to theinner centromeric chromatin are shown at the right.
teolysis of one or more centromere proteins is an obvious pos- as a knockdown of CENP-A also compromised KLN2 locali- At least two proteins are essential for CENP-A loading: In summary, even though recent experiments have yielded Mis18 and HsKNL2 hMis18 localises to centro- novel information about the timely recruitment of CENP-A meres in G2 but is removed as the cells enter mitosis. During to centromeres and a few critical components have been identi- mitotic exit it is recruited back to the centromeres at the same fied for this process, further work is required to understand the time when CENP-A is incorporated. Lack of Mis18 causes not priming step that happens during mitosis and is required for only loss of CENP-A from the centromeres but also an in- new CENP-A assembly. Furthermore, these experiments raise crease in acetylation of centromeric histones H3 and H4. This the surprising possibility that (1) either kinetochores function observation suggests that chromatin modifications are part of in mitosis with half of their CENP-A binding sites unoccupied; the priming event for CENP-A deposition. KLN2 (also known or (2) CENP-A binding sites are created during mitotic exit as Mis18BP1) has a myb motif that identifies it as a likely either by chromatin modification (presumably acetylation) or DNA-binding protein In mammalian cells, KNL2/ by proteolysis of a yet-to-be-identified substrate.
Mis18BP1 localises to the centromere during mitotic exit (lateanaphase–telophase and early G1) and its depletion abolishesthe localisation of CENP-A. Its function is conserved in C. ele- 4. What defines boundaries for the centromeric chromatin? gans although its localisation at centromeres is not restricted toa specific window of mitosis. CENP-A and KLN2/Mis18BP1 Several different histone modifications have been discovered are mutually interdependent for their centromere localisation, as being limited, enriched or interspersed relative to the centro- P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 meric chromatin. One of these classical modifications is the that co-operates together with the chromosomal passenger tri-methylation of Histone H3 on Lysine 9 (H3K9Me3). In protein TD60 to recruit the chromosomal passenger complex vertebrates, H3K9Me3 is constrained to the inner centromeric (CPC) to the centromere . However, the relative position- chromatin and . In the fission yeast S. pombe, peri- ing of H3K4Me2 and H3T3ph within the three-dimensional centromeric heterochromatin is enriched in H3K9Me2 where structure of the inner centromeric region remain to be deter- it forms a binding site for Swi6 (the fission yeast homologue of HP1) , which in its turn recruits the cohesin complex Despite numerous outstanding questions, one picture clearly HP1 and the cohesin complex localise in the inner centro- emerges. The vertebrate centromere, although it contains meric chromatin in vertebrates and . It is there- rather homogeneous DNA sequence elements that form its fore possible that H3K9Me3 in this case could have the foundation, is built from distinct domains The inter- same recruitment function as does H3K9Me2 in yeast. Yeasts nal (inner centromere) domain is dedicated to the control of also exhibit an enrichment of H3K9Me3 in pericentromeric sister chromatid cohesion (factors include cohesin and PICH chromatin but this modification does not seem to have a cru- ) and to the regulation of chromosome dynamics (chromo- cial role in centromere formation or function.
somal passenger proteins), while the outer region containing In humans and Drosophila, analysis of extended chromatin CENP-A is dedicated to directing the assembly of the kineto- fibres suggests that H3K4Me2 is interspersed with the chore itself. These two sub-domains are apparently specified by CENP-A-containing nucleosomes. However, this modification different histone codes that can act as stable epigenetic markers is detectable at centromeres in conventional mitotic chromo- for the recruitment of critical components for centromeric some preparations only in a minority of chromosome spreads function and assembly ). We hypothesize that chromatin and D. The function of such a euchromatic modifi- folding within the outer centromere is also an important key cation within the centromeric chromatin is not clear. It has for the maintenance of a special topology. Data from a num- been proposed that the alternating arrangement of CENP-A ber of groups suggest that inner centromere chromatin and and H3K4Me2-containing nucleosomes dictates a code speci- CENP-A chromatin have different biophysical properties fying centromeric chromatin .
that allow them to make different contributions to Another distinct modification of inner centromeric chroma- chromosome dynamics during mitosis tin is the phosphorylation of histone H3 at threonine 3 In Drosophila, heterochromatin is thought to prevent the This mitosis-specific modification is spread of CENP-A onto noncentromeric DNA and balanced generated by Haspin kinase and has recently been sug- levels of euchromatic and heterochromatic domains are gested to have an important role in chromosome segregation achieved by different histone-modifying enzymes and chroma- H3 phosphorylation on T3 appears to be a priming event tin components In S. pombe, barriers to heterochromatin Fig. 4. Centromeric chromatin organisation: possible models. (A) Model 1. Kinetochore chromatin is organised in a discontinuous array of DNAregions enriched for CENP-A and regions devoid of CENP-A. ChIP on CHIP analysis from the lab of Peter Warburton suggests that 2–3nucleosomes containing CENP-A are clustered next to a nucleosome which is CENP-A negative (B) This arrangement would produce a 30 nmchromatin fibre in which each turn contains CENP-A unless the periodicity differs from region to region. (C) Model 2. Based on fiber-FISH analysis,CENP-A containing chromatin is interspersed with chromatin containing H3K4Me2. This array is then flanked by a stretch of chromatin enriched inH3K9Me3 . Based on the limitations of the technique, which produces fibres of unknown axial compaction that not homogeneous in size, it isdifficult to determine the exact level of chromatin organisation to which these observations relate. (D) Model 3. CENP-A containing chromatin maypreferentially fold on itself, generating a distinct domain at the surface of the centromere. Stabilisation of the CENP-A domain could be either theresult of the unusual biophysical properties of CENP-A nucleosomes , or the presence of ‘‘stabilising factors'' recruited by CENP-A to the innerkinetochore.
P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 are defined by tRNA genes and require bound transcrip- CENP-A assembly . The balance between these antagonis- tion factors and RNA polymerase III to function In tic activities of CENP-B: CENP-A deposition and heterochro- humans, centromere chromatin and constitutive heterochro- matin formation, appears to be critical in determining whether matin also apparently exist as distinct domains that are sepa- exogenous a-satellite DNA will form a de novo kinetochore or a block of silent heterochromatin. This leads to the interesting, H3K9Me2. CENP-A mild over-expression causes a decrease though difficult to test, hypothesis that when de novo centro- in the amount of H3K9Me2 immediately flanking the centric mere activation or inactivation occurs using endogenous se- chromatin but does not change the level of H3K4Me2 quences, CENP-B should have a critical role in determining It therefore appears that CENP-A can spread along a a-satel- the outcome. For example, chromosome rearrangements pro- lite array, displacing H3K9Me2 but not H3K9Me3.
ducing dicentric chromosomes, might not undergo centromere To date, detailed studies of the functional relevance of cen- inactivation in the absence of CENP-B.
tromeric-specific histone modifications in vertebrates have All of the above leave us with a further puzzle to solve. How been mainly restricted to descriptive approaches. These are do cells activate a neo-centromere on a DNA region other than limited by the fact that drastic changes to the centromeric a-satellite where CENP-B boxes are lacking? Indeed, neocen- chromatin status would cause cell death. Only recently has a tromeres, like the human Y chromosome, lack associated novel approach yielded a HAC with a synthetic kinetochore CENP-B ), and thus their initial formation is almost cer- whose chromatin can be successfully manipulated and specifi- tainly CENP-B independent. However, neocentromere DNA cally inactivated in vivo making it the first conditional lacking CENP-B boxes is unable to nucleate de novo kineto- centromere outside of budding yeast This new synthetic chore assembly in human cells .
kinetochore contains regular repeats of a synthetic dimeric a-satellite DNA repeat, with one monomer having a tetracyclinerepressor (tetR) binding site (tetO) in place of the CENP-B 6. Rigid, plastic or elastic? box. Using tetR fusion proteins it is possible to target any pro-tein of interest into the synthetic kinetochore in vivo and assess Once the centromere is established and has directed the the subsequent chromatin changes and their consequence for assembly of a kinetochore, it exerts its main functions during kinetochore function Using this system, it was found that cell division. In mitosis, centromeres have essential signalling either ‘‘opening'' or ‘‘closing'' of the centromeric chromatin functions (we refer the reader to other reviews on this subject (by recruiting the tTA transcriptional activator or the tTS – Centromeres also have important mechanical/bio- transcriptional silencer, respectively) results in kinetochore physical properties which play a key role in the generation of inactivation. The binding of a transcriptional silencer caused an equilibrium of forces during chromosome alignment and the loss of CENP-A and H3K4Me2 accompanied by accumu- lation of histone H3K9Me3 and recruitment of HP1. These re- During chromosome bi-orientation at prometaphase, sister sults indicate that a balance between the different subtypes of kinetochores are attached to spindle fibres emanating from centromeric chromatin is essential for vertebrate kinetochore opposite poles. The pulling forces generated on the paired opposing sister kinetochores are counterbalanced by the resis- This new experimental system should allow a detailed anal- tance provided by the centromere. Thus, it is within the inner ysis of the molecular events which are temporally and spatially centromere chromatin that spindle tension exerts its effect and required for the maintenance of centromeric function.
is sensed by an as-yet unknown mechanism. What are the bio-physical properties of the centromeric chromatin and what isthe role (if any) of the different domains of the centromeric 5. How to make a centromere de novo The stretching of inner centromere chromatin is manifested From the early studies of human artificial chromosomes as sister kinetochore ‘‘breathing'' when chromosomes are (HACs), it emerged that de novo centromere formation from aligned and bi-oriented at metaphase Microtubule-depen- cloned alphoid DNA required the presence of CENP-B boxes dent stretching of chromatin in bi-oriented chromatids has . However, this was difficult to reconcile with the surpris- been known for many years but the compliant element that ing news that mice lacking CENP-B were viable and fertile regulates this intrinsic chromatid response is still a mystery . In order to rationalise this apparent paradox it was . Existing data reveal that the centromeric chromatin tempting to speculate that CENP-B is important for the estab- can be deformed. Are these deformations plastic or elastic and lishment of a centromere but becomes dispensable for its main- which kind of centromeric chromatin is involved? tenance in normal circumstances. In fact, it is only recently Recent studies in budding yeast confirm that the centromeric (almost 10 years after the initial studies) that it has been pos- chromatin is elastic. Upon depletion of H3 and H4 the com- sible to show that cloned a-satellite DNA arrays with wild- paction of pericentric chromatin decreased, so that sister kine- type CENP-B boxes are not competent for de novo centromere tochores were stretched further apart when chromosomes were formation and CENP-A assembly in cells lacking CENP-B bi-oriented. Surprisingly, this did not change the amplitude of (CENP-B/) What does CENP-B do? the typical mitotic oscillations . Therefore, the nature of the The recent studies reveal that CENP-B has a dual role, on determinants of the elastic behaviour of centromeric chroma- the one hand recruiting CENP-A to the chromatin during de tin remains elusive.
novo centromere formation, and on the other actively enhanc- Centromere elasticity has the curious property of being vec- ing the H3K9Me3 modification of chromatin containing chro- torial. When sister kinetochores are pulled in opposite orienta- mosomally-integrated a-satellite DNA without stimulating tions, the chromatin between them is clearly elastic, as P. Vagnarelli et al. / FEBS Letters 582 (2008) 1950–1959 described above. However, if the chromatin linking sister kine- [18] Mitchell, A.R., Gosden, J.R. and Miller, D.A. (1985) A cloned tochores is bent into a U-shape so that both kinetochores face sequence, p82H, of the alphoid repeated DNA family found atthe centromeres of all human chromosomes. Chromosoma 92, the same spindle pole, it is unable to straighten itself by elastic recoil unless pulled by spindle forces . Thus, kinetochores [19] Ikeno, M., Masumoto, H. and Okazaki, T. (1994) Distribution are axially elastic but radially plastic. How the chromatin of CENP-B boxes reflected in CREST centromere antigenic sites fibers pack within and between kinetochores to give the centro- on long-range alpha-satellite DNA arrays of human chromo- mere these remarkable properties is only one of many fascinat- some 21. Hum. Mol. Genet. 3, 1245–1257.
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