Summary The mammalian centromere is a multifunctional chromosomal domain with a complexity that is reflected in its higher order structure, DNA sequence organization and protein composition. The centromere plays a major role during cell division where it functions as the site for the integration of the chromosome with the mitotic spindle, the site of the mechanochemical motor responsible for the movement of chromosomes and the major and last point of interaction between sister chromatids. Recent studies have focused on characterizing the components of the centromere and establishing their relationship to its function. The following brief review summarizes some selected aspects of this recent work. Introduction: The Higher Order Organization of the Centromere The mammalian centromere (primary constriction) appears as a distinct structural domain within the metaphase chromosome (Fig. 1).The manner in which chromatin is packaged within the metaphase centro-
mere can be understood at least in a broad sense within the context of current models of chromatin higher order structure. In gencral. chromatin in the form of contiguous loop domains is packaged in a series of steps to form a 250nm fiber. This fiber is further packaged, through one level of coiling, to form the arms of the metaphase This last packaging step does not occur in thc centromere, hence the 250nm fiber represents the final stage in the condensation of the metaphase centromere('1. Clues to the higher order organization of the interphasc centromere have been obtained from studies of the AT-rich centromeres of the mouse. Mus rnuhculus. These centromeres are sensitive to the drug 33258 Hoechst and 5-azacytidine. Growth in the presence of either of these agents results in an interference with the normal condenbation of the centromere during the interphase-mitotic transition. Thus, chromosomes from treated cells have centromeres that have reduced diamctcrs (-100nm) and are at least five times their normal metaphase length. Recovcry from drug treatment results in the formation of centroineres with normal morphology through a process that includes coilin and condensation of the extended centromere fibcr'a. This process may mimic in vivo changes in centromere organization that occur during the interphase-mitotic transition. Thus a moderately relaxed form of the 250nm fiber may represent the predominate configuration of the interphase centromere. In addition. the packaging process observed in drug recovery suggests that coiling inay play a role in the formation of the 250 nm fiber as well as the further packaging of this fiber in the chromosomal arms. These various levels of coiling can frequently be observed in spread preparations of pro-metaphase chromosomes from untreated cells (Fig. 2 A,R) and are summarized in Fig. 3A.
Fig. 1. Scanning electron micrograph o f chromosomc 1 of the Indian munt,iac. The centromere region (C) can be seen as a constriction along thc chromosome. The kinetochore (K) appears as a surface specialization at the centromere. Fig. 2. ( A ) Chromosome 1 o f the Indian inuntjac from an acetic acid/methanol spread preparation of a pro-metaphase cell. The cliromosome is composed of ii series of coils of a 25Onm fiber. The centromere region shown at high magnification in (B) is formcd by a smaller fiber that is also coiled (C-centromere). A completely condensed chromosomc 1 of the Indian munljac is illustrated in (C). Autoantibodies react with either thc ccntral domain (D) or the kinetochore domain (E). (F) illustrates the pattern produced by antibodies to the CLiP antigens. The antigcns are located at discrete sites along the pairing surface (small arrows) as well as along a filament that connects sister kinetochores (largc arrow).
._ -_--. -.
Pairing domain Central domain
Fibrous corona Outer plate Middle space
I Inner plate Chromatin
Fig. 3. (A) The higher order structure of the centromcrc. A fully condensed metaphase chromosome is shown in A. The path of the 250nm fiber in the centromere region (bracketed area) and arms o f the chromosomes are illustrated in B. The coiled path of the -100nm subfiber is shown in C and the expanded form of the centromere found in chromosomes grown in either 33258 Hoechst or 5-azacytidine is shown in D. (B) The domain organization of thc ccntromere.
There may be several reasons why the higher order structui-e of the centromere is distinct from the rest of the chromosome. First, an uninterrupted expanse of the 250nm fiber may be a requirement for the proper configuration of the kinetochore. Secondly, this expanse may facilitate the interaction between sister chromatids at the inner surface of the centromere. The importance of the three-dimensional configuration of the centromere to its function is reflected in the fact that the 250 nm fiber is not structurally or compositionally homogeneous at the centromere. Rather, the fiber can be subdivided into at least three distinct structural/ functional domains: the kinetochore domain along the outer surface of the centromere, the central domain representing the majority of the centromere and the pairin domain at the inner surface of the centromere(E5) (Fig. 2 D,E,F). Although specific functions can be assigned to each of these domains, they do not function in isolation. Rather it is their integrated function that ensures orderly chromosome segregation. The domain organization of the centromere is summarized in Fig. 3B.
The Kinetochore Domain Ultrastructurally the mammalian kinetochore appears as a specialization along the outer surface of the centromere and consists of three regions: (1) an inner plate overlying the central domain: (2) a middle electron translucent zone; and ( 3 ) an outer plate. A fourth area called the fibrous corona is found covering the outer plate when microtubules are absent (Fig. 3B). In this review, I shall consider the kinetochore domain to include this trilaminar kinetochore structure, the chromatin that is subadjacent to the inner plate, as well as the chromatin that imrncdiately surrounds the lateral margins of the kinetochore plates. 1 have taken this
view because the region surrounding the kinetochore plates appears to be functionally related to the kinetochore. For example, microtubules have been found to terminate at the inner plate, outer plate, and to interact along the fibrous corona (e.g. refs 5-7). Similarly the chromatin adjacent to the kinetochore plates also acts as a site of microtubule interaction('). Evidence for DNA at the kinetochore includes: (1) The direct observation of an interaction between chromatin fibers and kinetochore [email protected]
); (2) the observation that DNase I decondenses the kinetochore plates('); and (3) the detection of a nuclease sensitive, phosphorous-rich 30 nm fiber within the layers of the kinetochoreC6).Unfortunately, attempts to identify DNA sequences that map specifically to the kinetochore domain or within the layers of the kinetochore in any mammalian chromosome have not met with success. One possible exception is the mousc minor satellite (see below)(''fi. DNA must play an important role in kinetochore organization either from a position underlying the structure, specifying placement, or within the structure, more directly determining structure. Therefore, the identification of kinctochore-related DNA sequences represents an important unfulfilled goal in our understanding of this centromere domain. The identification of autoimmune sera that react with the centromere(ll) ignited the hope that it would be possible to identify proteins unique to the kinetochore domain and in particular the kinetochore plates. It was expected that kinetochore domain proteins might fall into two broad classes; those that played a primary role in forming the structure of the kinetochore layers or the surrounding region, and those that were related more directly to the function of the kinetochore. I will consider the domain assignments of all identified centromere proteins together, in the following section.
assigning DNA sequences to this region in a wide variety of species. In general, the central domain of mammalian centromeres contains repetitive sequences. For example, S % of the human genome is composed of the alphoid family of satellite DNA sequences and they are localized to all the centromeres of the human karyotype(17). An alphoid subclass has been shown to overlap perfectly with the sites of anti-centromere antibody binding. This subclass contains a 17bp motif (S'CTTCGTTGGAAACGGGA3') that has the ability to bind the centromere protein CENP-B and this motif has been termed the CENP-B box("). Perhaps the best documentation of the DNA scquence distribution within a mammalian centromere at present is found in the mouse, Mus mitscuZus. This species contains two prominent satellite classes, the major satellite (5-10% of the genome) present at all the centromeres of the karyotype except the Y and the minor satellite which has the same karyotype distribution("). Because it is possible to obtain expanded centromeres in the mouse, with either 33258 Hoechst or 5-azacytidine, it has been possible to obtain detailed information concerning the distribution of satellite Fig. 4. An Indiaii inuntjac metdphase cell reacted with an antibody to ~ 3 4 ' ~ "The ~ . antibody reacts with the centrosome (c). kinetochorc sequences within the centromere. Interestingly, the microtubulcs and the kinetochorcs (small arrows). In contrast, major satellite appears to be distributed uniformly antibodies to the glycolytic enzyme enolasc ( 5 )react only with the throughout the centromere, representing a major centrosome .(c) and the kinetochores (small arrows). Thus, component of the central domain; while the minor kinetochore-associated proteins may have different distributions satellite is confined to the region of the kinetochore within the mitotic apparatus. domain(") (Fig. 5A to E). Major satellite DNA is ATrich and contains a curvature near the 3' end of the Generally, however it has not been possible to 234 bp monomer('9). The alleviation of this curvature document that any centromere protein identified thus with the drug distamycin A interferes with the far plays a direct role in determining the structure of the condensation of the centromere. Thus, at least in kinetochore domain. In contrast, there is a growing list mouse DNA, conformation may play a direct role in of kinetochore-associated proteins that are likely to be determining central domain higher order structure. A involved in the function of the kinetochore domain. A motif similar to the human 17bp CENP-B box is major characteristic of these proteins is that they are not present within the minor satellite but is not found in the detected in association with the interphase centromere major satellite(lo318)).Thus the distribution of the but are found at the kinetochore only during cell CENP-B box in M w musculus is confined to the region division and are previously identified cellular comof the kinetochore domain whereas in human it is ponents. Interestingly, many of these proteins are also probably present predominantly within the central found at other microtubule organizing centers, includdomain judged from the distribution of the protein ing the centrosome and midbody. and some a;e also CENP-B") (see below). The unique cytological localizassociated with the kinetochore microtubules("). It is ation of the minor satellite in Mus musculus makes it an beginning to appear that microtubule organizing ideal probe for the future investigation of the DNA centers, including the kinetochore domain have the composition of the kinetochore domain in this species. ability to act a5 sinks for ubiquitous cellular proteins. So far, a number of both repetitive and unique DNAs Identified kinetochore-associated proteins include tubulin(l3), calmodulin("). dynein(") and p34cdc2(16) have been mapped adjacent to the minor satellite (Wong and Rattner, unpublished observations). (Fig. 4A). In addition we have found that antibodies to As previously indicated there has been a major the glycolytic enzymes enolase (Fig. 4B) react with the attempt to identify domain-specific centromere proteins kinetochore domain (unpublished observations). This using autoantibodies. The apparent molecular weights reactivity may indicate that there is a spatial link of putative centromere proteins, as determined by between energy production and the mechanochemical immunoblots to human proteins, vary between reports motor responsible for chromosome movement. but ma be grouped into cix classes based on molecular weightXo).The most discussed centromere proteins are The Central Domain a group of three autoantigens that have been designated The central domain represents the bulk of the area of CENP-A (17 kd), B (80 kd), and C (140 kd)("). CENPthe centromere and progress has been made in A has only recently been isolated and characterized and ~
Fig. 5 . A mouse metaphase chromosome from the cell line L929 (A). Cells grown i n the prescnces of 33258 Hoechst have chromosomes with uncondenscd centromeres (B: arlows). I n situ hybridization indicates that the major satellite of mouse is located throughout the centromere in these chromosomez (C) while the minor satellite is confined to a small defined domain (D.E) that cci-localizes with thc kinetochore domain.
has proved to be a centromere-specific histone(22)with some evolutionary relationships to histone H3 (Palmer, O‘Day, Le Trong, Charbonneau and Margolis, personal communication). Human CENP-B has to date been the best characterized of the CENP antigens. The sequence of its cDNA has been obtained and monospecific antibodies are available(’). The C-terminal region of the protein contains two acidic groups that may be central to the function of this protein, and CENP-B has been shown to be spatially associated with t~bulin(’~).The distribution of this protein in human chromosomes has been determined by immunoelectron microscopy and it appears to be distributed largely within the central domain(“24).The amount of CENP-B varies between chromosomes of the human karyotype, in contrast to CENP-C, which appears to be equally distributed betwcen chromosomes. At present CENP-C remains uncharacterized. Microinjcction studies with antibodies suggest that the CENP antigens are involved in interphase events that are necessary for proper centromere function during mitosis(252-61 Autoimmune antisera that include antibodies to the CENP antigens have been applied to chromosomes from a variety of mammalian species and in each case these sera produce centromere reactivity. The use of polyclonal sera together with the small size of the centromere region of most mammalian chromosomes has, in most cases, left it unclear which antigen(s) is responsible for centromere reactivity and in which domain the antigen(s) resides. However, the following specific information is known. (1) Sera containing antibodies to centromeric proteins including CENP-A, B and C react primarily with the kinetochore domain in rat kangaroo (PtK’) chromosomes as determined by immunoelectron microscopy(”). (2) Sera containing antibodies to centromeric proteins including CENP-A, B and C selectively react with the kinetochore domain of the large chromosomes of the Indian muntjac wherc the three centromere domains can be distinguished at the light microscope level(20,’s). Monoclonal antibodies to human CENP-B do not, however, react with the muntjac kinetochore domain (Earnshaw and Rattner, unplublished observations). ( 3 ) When sera from a large number of autoimmune paticnts with anti-centromere
autoantibodies were reacted with Indian muntjac chromosomes, the majority reacted only with thc kinetochore domain while a small ercentage reacted with the central and pairing domainsr2”). (4) An affinitypurified antibody to the 50 kd centromere protein, CENP-D, showed specific reactivity with the kinetochore domain of Indian muntjac chromosomes(”). CENP-D has also been idcntified in western blots of rat chromosomal proteins and may be similar to the mitosis-specific autoantigen detected by Hadlaczky and ~ o - w o r k e r s ( ~ ~Thus, 3 ~ ~ )a. majority of human autoimmune sera contain anti-centromere autoantibodies that can recognize epitopes that reside specifically within the kinetochore domain of a variety of mammalian species. The question of whether specific centromere proteins are widely conserved within mammals has bccn addressed in a limited number of studies. A band corresponding to the position of CENP-A has been identified on immunoblots of human, mouse, Indian muntjac, swine, hamster, rabbit, Chinese hamster and cow cellular extracts using human autoimmune sera(zo”o,”) . A putative CENP-B and CENP-C band has been detccted in immunoblots of mouse and a band corresponding to CENP-D has been identified in mouse, human, Chinese hamster, rat and Indian muntjac(20,21,30and Rattner unpublished ohseivationc) ~1~~ cDNA for CENP-B of the mouse, Mus muscufus has recently been obtained.. Comparison of the human and mouse cDNAs reveals a 96% homology within the coding region (Kevin Sullivan, manuscript in preparation; Cusano and Rattner, unpublished observations). This finding is in agreement with the conserved nature of the CENP-B box between human and mouse. Southern analysis using the CENP-B box motif as a probe, however, suggests that this motif is not conserved throughout the mammals or within a genus or species (Wong and Rattner, manuscript in preparation). For example, this motif was detected in gorilla and chimpanzee but not in the more primitive primate, the African green monkey. Within the genus M u s , the motif is present in Mus niusculus and Mus spreius but not the ancestral species Mus caroli. Similarily the minor satellite is present in Mus mrcsczllirs and MLLS spretus but not in Mus These observations would seem to suggest that the association between thc
CENP-B box as it is presently defined and CENP-B may be a feature of more recently evolved species. Interestingly, in Mus sIiretus the minor satellite is not confined to the region of the kinetochore as is the case with the closely related species MUJ musculu, but is present throughout the central domain in a wa similar to that found in the human k a r y ~ t y p e 1 l ).~ ~It. ~is tempting to speculate that the CENP-B box was first distributed throughout the centromere and that its localization to the kinetochore region is a recent evolutionary event in some species. This would explain the differences in the apparent domain distribution of CENP-B between mouse and man previously noted in this review. It is tempting to speculate that, like CENPB, the domain assignment of some other conserved centromere proteins will also be found to be speciesbpecific. However, it seems reasonable to suspect that conserved proteins that play a major role in the organization and/or function of each of the centromere domains will have retained their domain assignment throughout evolution. The CENP antigens are associated with the ccntromere throughout the cell cycle. However, other centromere proteins have been idcntified that associate with the centromere only at cell division(29). In addition, some proteins are present throughout the chromosome, including the [email protected]
),or are found transiently associatcd with the centromere(2s”7). The Pairing Domain The region along the inner surface of the centromere that represents the site of interaction between sister chromatids at metaphase has been termed the pairing domain. In general, two classes of proteins have been assigned to this domain. The first, the INCENP (INner CENtromere Proteins) are found at specific sites along the points of sister chromatid association(”’). Two polypeptides of 135 and 155kd have been identified in chicken and a 140kd protein appears to be their counterpart in human. Another class of pairing domain proteins are the CLiPs (Chromatid Linking Proteins)(”). These proteins, detected with human autoimmune sera, are localized to a filamentous structure that transects the central domain and extends between sister kinetochores as well as within a group of sites along the inner surface of the centromere and along the arm5 where sister chromatid contact occurs (Fig. 2F). Following chromatid separation the INCENP proteins migrate to the equatorial zone of the spindle whereas the CLiPs are no longer detected. The detection of specific foci for proteins along the inner surface of the chromatid and a structure that interconnects sister kinetochores represents the first step in uncovering the manner in which chromatid disjunction is controlled. In conclusion, there is a growing consensus concerning the basic organization of the mammalian centromere. While our understanding of this structure still lags behind that of simpler organisms, in particular the
yeasts(34),there is reason to hope that we will soon be able to establish compositional-functional correlates for the mammalian centromere. Acknowledgements Appreciation is extended to Alex Wong, W. C. Earnshaw. B. R. Brinkley, Kevin Sullivan, K. D. Palmer, K. O‘Day, H. Le Trong, H. Carbonneau and R. L. Margolis for sharing unpublished information and providing helpful comments during the preparation of the manuscript. References 1 RATTNER. J. B.
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