The centromeres of the Indian muntjac: Evidence for the existence of multiple centromeres?

ELSEVIER Mutation Research 356 ( 1996) 187- 195

Fundamental and Molecular Mechanisms of Mutagenesis

The centromeres of the Indian muntjac: Evidence for the existence of multiple centromeres?

Derek R. Latour a, Baldev K. Vig a.*, Eva-Maria Finze b, Neidhard Paweletz b ’ Department of Biology /314< University of Nevada, Rem, NV 89557-0015, USA

b Research program IV, Division 0430, German Cancer Research Center, Im Neuenheimer Feld 280. D-69120 Heidelberg, FRG

Received 7 December 1995: revised 6 February 1996; accepted 22 February 1996

Abstract

Unlike the centromeres of other species, the ‘compound’ centromeres of the Indian muntjac span over exceptionally extended regions (Brinkley et al., 1984). We extend this concept and show that some of these centromeres are divisible into several chromomeres in which the light staining regions alternate with the dark staining C-band positive segments. Unlike the centromeres of other species where the centromere replicates as one unit, the replication of the sub-units constituting the centromere of the X-chromosome in the muntjac occurs at different times as at least three independent segments. The CREST staining of the centromere regions of even the smallest (Y,) chromosome is interrupted by non-staining segments. Electron microscopy shows similar interruptions in the continuity of the trilamellar kinetochore. Sister chromatid exchanges occur in the region of the centromeres and chromatid breaks within the centromere region occur in the non-fluorescent segments. We interpret these data to suggest that the centromere regions of the Indian muntjac are made up of independent multiple centromeres interrupted by non-centromeric chromatin. Relevance of these parameters in mutagenesis is briefly discussed.

Keywords: Centromere; Indian muntjac; Multicentric; Multiple centromere

1. Introduction

Generally, the centromere of a chromosome is a specific ‘pin-point’ structure which stains lightly with basic dyes or remains unstained. However, the centromeres of at least one mammal, the Indian muntjac (Muntiacus muntjac uaginalis, 2n = 6 fe- male, 7 male), are several microns in length. The

* Corresponding author. Tel.: (702) 784-6544: Fax: (702) 784-

1302.

size of the centromere of the X-chromosome in this species is larger than the smallest chromosome in the genome, the Y2. The centromeres of the Indian muntjac have been referred to as compound centromeres (Brinkley et al., 1984) implying that more than one centromere ‘subunits’ are juxtaposed end- to-end to make the length of the centromere. The proposed model suggests that during the evolution of the compound kinetochore any non-centric chromatin was entirely eliminated. It has even been proposed that any centromere is a structure made up of multiple repeats of several subunits. These units

0027-5107/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SOO37-5 107(96)00053-X

are suggested to be compoxed of DNA xegmcnth

which harbor alternating sites of reaction to the scleroderma serum with tho\c which do not

(Zinkowski et al.. 1991). Thus. even though the entire region is suggested to be made LIP 01’ cjnc centromere without interruption by non-ccntromeric chromatin, electron microscopy of \ome of these ‘centromeres’ exhibits a gap in the continuity 01‘ the trilamellar kinetochore. The nature and significance of the region representing the gap haa so far been over-looked.

The centromeres of the Indian muntjac react quite well to the antikinetochore antibodies present in the sera of scleroderma (var. CREST) patients. How- ever, it is not known if the reaction represents ;I specific centromere protein(s) (Earnshaw and Roth- field, 1985). However. the reaction to antikinetochore antibody show\ ‘non-staining’ gaps in cvcry centromere of the muntjac ~ a situation which co11- trasts with the single-unit centromere structure observed in the related Chinese muntjac (M~ltic,~r.\ rcw~csiae; 2~1 = 46) (Brinkley et al.. 1984). A sim- lar situation is encountered when the chromosomes of the two species are labeled with the telomere probes (Lee et al.. 1993). Every chromosome of the

Indian muntjac. including the centromere region in some. exhibits multiple sites of hybridization. The chromosomes of the Chinese muntjac show only two sites at the termini of each chromosome.

It has been proposed that the centromeres ot‘ the Indian muntjac are composed of a series of tandcmly fused centromeres of the Chinese muntjac. The term ‘compound centromere’ implies that the rejoining of‘ the unit centromeres some how eliminated the partic- ipation of any non-centromeric DNA in the proccsk of translocations which created the long linear nrck- like centromeres of the Indian muntjac. In this con- munication we suggest that these centromerrs art‘ not composed of repeat sub-units but. at least in the case of X-chromosome. are made up of a minimum of three individual centromeres which are interrupted by non-centromeric chromatin. The support for this concept is provided by linear differentiation oh- served within the centromeres using various techniques: the discontinuous replication of the suhre- gions of the centromere. the occurrence of somatic crossing over within the centromere of each chromosome and the presence of chromatid breaks only in

the inter-centromeric regions in these multicentric structures.

2. Materials and methods

The Indian muntjac cells line. ATCC cat. no. CCL 157. derived from a male was employed in this study. The cells were grown without CO, in McCoy’s 5A supplemented with 392 mg/l of glutamine. IO0

units each of penicillin/streptomycin mixture and IS’% fetal calf serum. Colcemid was applied thy ft- I2 II before harvest. The cells were fixed in 3 : 1 methanol/acetic acid mixture and were used l’ol routine Giemsa staining, G-banding or C-banding. In an attempt to induce decondensation of the ccn- tromere region, the cells were also treated with Iinrchst 33258. 5-bromodroxyuridine (BrdU) or 5- azacytidine. each with IO mi M for I8 and 7-l h. For study of DNA replication, BrdU (10Y5 M) was applied for various intervals (see results) before hat-- vest. BrdU was detected using antiBrdU antibody

(Sigma) using the protocol provided by the manufac- turer.

The putative sites of kinetochores and their morphology was studied by applying antikinetochorc antibodies present in the serum of a scleroderma (var. CREST) patient (designated SH). by purified anti-CENP-A and monospecies anti-CENP-B and C. The technique for the serum reaction is given else- where (Vig et al.. 1994). The reaction to anti CENP.. .4. anti CENP-B and anti CENP-C was studied using minor modifications of the same techniques. The details of preparation of these antibodies are given in Palmer et al. ( 1991 1 for CENP-A, Cooke et al. ( IWO) for CENP-B and Saitoh et al. ( 1993-) for CENP-C. The trilamellar structure of the kinetochore WI\ studied using electron microscopy which was

performed using the technique already outlined (Paweletz et al.. 1989).

Increase in the frequency of‘ sister chromatid es- changes was accomplished by exposing the ceils to various concentrations of mitomycin C (MMC) and N-methylnitronitrosoguanidine (MNNG). Since our intention was to study these exchanges only in the crntromere region, the detection and analysis was accomplished by tagging the cells with the CREST \cruni.

D.R. Latour et al./Mutation Research 356 (1996) 187-195 189

3. Results

3.1. Morphology of the centromere region

The application of Hoechst or Sazacytidine did not affect the condensation pattern of the centromere region; BrdU was only modestly effective. However, in the BrdU treated cells, careful observation of the region showed that the centromeres are divisible into seven sub-regions (Fig. la). Occasionally, upon sim- ple Giemsa staining a fully extended metaphase centromere of the X chromosome could also be divisible into three lightly staining segments alternating with four darkly staining regions. Upon C-banding the untreated chromosomes exhibit two rather large and one smaller block of C-bands (Fig. lb). G-banding reveals two bands within the centromere of the X- chromosome but only one band in the region of chromosome 1 (Fig. lc). This differentiation clearly shows a lack of uniformity within the centromere - a fact in contradiction to the structure of the centromeres of other species. It can be interpreted that the centromere region of the X chromosome is a multicentric structure in which the individual centromeres are interrupted by non-centric chromatin.

We suggest that there are at least three separate

centromeres represented by the light regions in the Giemsa stained centromere of the X chromosome.

3.2. The reaction of the CREST serum and anti CENP antibodies

The interphase cells show a series of many small signals of unequal dimensions. The number varies from cell to cell and is not easily countable. We attribute the discontinuity of the signals to the presence of non-centromeric DNA. This DNA may be small blocks of heterochromatin, telomere sequences or other non-centromeric sequences. On metaphase chromosomes, the reaction to the CREST serum was not uniform, either. The X chromosome exhibited

three segments of fluorescent tag interrupted by two non-staining regions. All other chromosomes showed at least two segments each; this pattern included the smallest Y2 chromosome also (Fig. 2a,b).

Even though the reaction to whole serum is known (Brinkley et al., 1984), we included this study to compare the reaction by the individual anti CENP- antibodies. We studied the chromosomes after react- ing with anti CENP-A, -B and -C. On metaphase

a b

Fig. 1. Indian muntjac cells showing linear differentiation in the centromere region, particularly that of the X-chromosome. (a) A BrdU

treated cell; each of the X-chromosome (x) in the two photographs shows four dark bands interrupted by three lightly staining regions. The

pair of chromosomes no.1 (arrows) shows two dark bands. (b) An untreated cell processed for C-banding. Note that the two peripheral bands

(large arrowheads) are heavier and darker than the one in the middle (small arrowhead) of the centromere of the X chromosome. These

bands do not relate to the darker regions in the centromere shown in (a). (c) An untreated G-banded cell shows two dark bands and three

light bands in the region of the centromere (double arrowheads). This banding pattern is reverse of the one seen in (b).

chromosomes. the pattern of reaction after anti- CENP-A treatment was similar to that observed with the whole serum. However, analysis of several number 1 chromosomes showed two to three sites of reaction. In some cases (see Fig. 3d). the sites on one chromatid were separated by considerable distance. Even the Y, chromosome showed two distinctly separate sites of reaction. We consider this as an indication of stretching of the CREST-negative chromatin interspersed between the centromeres. Anti- CENP-B did not exhibit reaction to any centromere of the muntjac even though the western blot (not

reproduced here) did identify a protein at approx. X0

kDa position. The reaction of antiCENP-C (Fig. 2e) was fainter than that of CENP-A but, never-the-less. was characteristic of the whole serum.

A point of interest in the CREST and other anti CENP antibody treated cells is the occasional occur-

rence of chromatid breaks (see inset in Fig. 2e). These breaks were always observed at the region which was negative to the reaction. Apparently, the position of breaks correspond to the dark staining segments in the centromere region after Giemsa staining, e.g., as seen in Fig. la. The frequency of these breaks was observed to increase after treatment

with clastogens like MNNG or MMC.

Fig. 2. Reactron of the Indian muntjac genomr to the CREST ~-urn (a-c). Note linear differentiation in the marked chromosomes X and I. each showing three fluorescent segments interrupted hy unstained gap”. Similar reaction of the cells to purified anti CENP-A (d) and

monospecies anti CENP-C (e) antibodies. Note the separatmn of the two signals on the chromosome marked Y, both in CREST treated (h)

and purified anti CENP-A treated (d) cells. Also note the occurrence of sister chromatid exchanges (small arrows) in some chromosomes in

the CREST treated cell (h. d). OccaGonal breah in the centromerr repion [inset m (e)] were seen to always occurs in the non-fluorescent part

of the multicentric structure.

Fig.

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At morphological level, our electron microscopic preparations of the centromere-kinetochore complex confirmed what light microscopy revealed. The thin as well as serial sections showed that every analyz- able centromere region is associated with at least two discontinuous trilamellar structures separated by

non-kinetochore regions. The X chromosomes showed three kinetochore units separated by two non-kinetochore gaps. Even the Y2 chromosome, the

smallest in the complement, showed two kinetochores placed next to each other (Fig. ?a). This observation confirms a relationship between the number of trilamellar kinetochores and the number of sites of reaction to the CREST serum. Our inter-

Table I

Frequency per cell of sister chromatid exchange5 in the cen-

tromere region of the Indian muntjac induced hy MMC and

MNNG. Two hundred cells were analyzed per treatment

Treatment Concentration

MMC

MNNG

0 (control) IO-’ M IO- ’ M IO i M

0.13 0.28 0.2 I 0.23

0.13 0.18 O.lY 0.13

Frequency per cell is rounded off to two decimal place

pretation of the serial sections, though similar in morphology to that of Brinkley et al. (1984). is that these sub-units are not connected to each other as previously thought.

Fig. 4. A series of three cells showing BrdU incorporation in exponentially growing cells for 7 continuous hours (a). 7.5 continuou\ hour\

(b) and the interval between 9-38 h (c) before harvest. In (a) the incorporation of BrdU is limited to a very small segment closest to the

short arm of the chromosome. The replication pattern in the cell shown in (b) is more pronounced in that an additional band is \isihlr.

During the interval 3-4 h into the S phase, the X chromosome replicates the segment adjacent to its long arm cc). (d) Diagrammatic

interpretation of the results to show that replication of the entlre centromere region does not occur as a single unit.

D.R. L.atour et al./Mutation Research 356 (1996) 187-195 193

3.4. Sister chromatid exchanges within the centromere region

The CREST treated cells showed sister chromatid exchanges within the fluorescent region of the centromere (Fig. 2b,c). The frequency of these exchanges was in the range of approximately one exchange per ten cells. It was not possible to un- equivocally state that these cross over-like configurations did not simply represent physical twisting of the two sister chromatids in the long region of the centromeres, particularly the X. This problem was addressed by exposing the cells to two chemicals which are know to increase the frequency of sister chromatid exchanges. The cells treated with MMC and MNNG (each at 10e5, 10e6 and lo-’ M concentration) exhibited an increase in the frequency of these exchanges (Table 1). We, therefore, conclude that these configurations represent sister chromatid exchanges originating, apparently, in the regions which do not react with the CREST serum, i.e., in the regions devoid of the centromeric chromatin. We Giemsa stained the cells after recording the position of sister chromatid exchanges. Whether these exchanges occur in the heterochromatin or not could not be determined precisely.

3.5. Separation of the centromere region

Centromeres of a given genome have been re-

ported to separate into two daughter subunit in a sequential manner. However, in all the species so far examined (e.g., mouse, man, cattle, Chinese hamster, broad bean), a given centromere splits into two units at once along its entire area. This is expected because of the minute size of the centromere. Individ- ual centromeres of the Indian muntjac, however, split into two units in a manner reminiscent of the sequential separation of several chromosomes in other genomes. The X chromosome, for example, generally separates into its units at the centromere located in the middle and proceeds on towards the two ends. When separation begins in the middle of the centromere, it may be followed by separation of the segment adjacent to the short arm or to the long arm. In brief, the centromere separation in these chromo-

somes is not an all or none phenomenon; a sequen-

tial pattern of separation occurs within a centromere. This observation further supports the notion that these centromeres are composed of more than one unit.

3.6. Replication of the centromere region

The centromeres generally show replication pattern which is compatible with a single unit of replication, i.e., the entire primary constriction either shows the label or the label is entirely absent. The centromere region of the X-chromosome of the In- dian muntjac, for example, does not follow this pattern. It replicates in segments separated by an interval of up to 2 h. When cells were fed with lo-’ M BrdU during the 6 h before harvest, no label was observed on the metaphase chromosomes. The first signs of label appeared when cell were fed BrdU continuously for 7 h before metaphase chromosomes were harvested (Fig. 4a). At this time, which coincides with the cell having been into the S phase for 5 h (Comings, 19711, the labeled segment is a small region at the junction of the short arm and the centromere. If BrdU is allowed to stay in the medium during the last 7.5 h, two replication bands are visible. One of these bands coincides with that observed at 5 h into the S phase and the new one is just below it. The two bands are separated by a gap or a brief region in which BrdU, if present, could not be detected. The third pattern of replication was observed when cells were labeled during the last 9 h of culturing, i.e., when the cells were into the S phase

from 3 h onward. However, the label in the 9 h treatment was too heavy to permit a rational conclu-

sion. We, therefore resorted to 1 h of pulse label between 9 and 8 h before harvest. This treatment produced two bands. One appeared to coincide with the band produced at 4.5 h into the S phase; the other rather heavy band spanned the region at the bottom of the neck and the long arm. This study might indicate that the centromere region of the X chromosome of Indian muntjac takes around 2 h to complete its replication and that the pattern of replication is a discontinuous pattern. An interpretation of the results is given in Fig. 4.

4. Discussion

The term ‘compound kinetochore’ hah been used to “describe large kinetochores like those seen on chromosomes of the Indian munt,jac and ‘unit kinetochore’ to identify .smaller conventional kinetochores that may have fused durin g evolution to produce

compound kinetochoreb” (Brinkley ct al.. 1984). Even though the term centromere and kinetochore have been used synonymously and the two or- ganelles go hand in hand. one must diftcrrntiatc between the trilamellar. proteinaceous structure (the kinetochore) which apparently provides a facultative overlay on the underlying DNA-protein complex at the primary constriction (the centromere). Thuh. in reference to the structural makeup of the primary constriction, the term compound centromere appears more appropriate than the term compound kinetochore.

We suggest that the region called the centromerr in the Indian muntjac may be referred to as a rlllr/ti- ~ntric region. These centromere regions seems to be composed of several individual centromeres separated by non-centromeric chromatin. This concept 01. multicentricity of a chromosomes in a true breeding organism is perhaps not new. One has to consider the genomes of the likes of butterflies, Luzula and the scorpions even though the centromeres in these or- ganisms are not present in a series of continuoux

juxtapositions. However. in a neoplastic cell line of rat endothelial origin, we reported a chromosome which carries at least six tandemly arranged ten-

tromeres interrupted by non-centric segments (Paweletz et al.. 1989). This chromosome appears to segregate in orderly manner.

We propose that the light staining segment:, between the dark staining regions (Fig. la) represent the real unit centromeres. Thus. thr wt~ttwwt-c of the X-chwinosof~~e is LI nllilti~~enttk re,yfo~l IIIL~/CJ up

of .rer~eml indqwrldent (‘elltl’ol)l~‘Iv.F. The presence of telomere sequences within the centromere region 01. the Indian muntjac (Lee et al.. 1993) further COIN- firms the presence of multiple crntromeres. Concur- ring with the arguments presented by Brinkley et al. (1984). the number of independent centromeres in these chromosomes may be more than the number- ot CREST positive sites or even the three site\ of trilamellar structures. However. light microscopy

does not permit separation. for example of the centromere of the X chromosome, into more than three

to four distinct segments. The staggered but more or less sequential separa-

tion of the region within a so-called centromere may he of interest in defining the multicentricity of these chromosomes. The previous reports of the ‘halo’-like opening in the centromere of the X-chromosome (Comings. 1971) can be explained by the sequential \eparation of individual centromeres. The separation

of the centromere in the middle ahead of the separation of other two centromeres would produce such a -bubble‘. The position of a centromere in the sequence of separation depends upon the quantity of heterochromatin (Vig, 1983). Since the middle centromere is associated with the smaller quantity of heterochromatin. this intrachromosomal pattern of separation appears to follow what is known for the interchromosomal pattern (Gerlach et al,, 1984).

Some segments of the centromere of the X-chromosome replicate as far as 2 h ahead of other

regions. This pattern testifies to the independence of these DNA segments in the timing of replication and suggests that these segments constitute separate replicons. Unlike the cells fed with tritiated thymi- dine (Comings. 1971) which does not permit precise localization of the replicating sites, no BrdU label was heen specifically over the intervening non-het- crochromatic regions which might constitute the true ccntromeres. This may be due to low A : T content or some other unknown reason. What ever the basis. the replication pattern is another factor supporting that these centromere regions are multicentric.

The localization of the various CENPs shows the hegmental nature of the centromere region: even the Y, chromosome shows two segments. This concept also finds support from the analysis of serial sections obtained through electron microscopy. Even though it has been long reported that the centromeres of the X-chromosome exhibit three distinct kinetochores. argument has been presented that these regions represent artificial segmentation or discontinuities (Brinkley et al.. 1984). We believe that this segmentation represents the real situation; we show that even the centromere region of the smallest chromo- \ome also exhibits two separate kinetochores.

The segmentation of the CREST reactive region. particularly in the interphase cells (Brinkley et al..

D.R. Latour et al./Mutation Research 356 (1996) 187-195 195

1984; Latour, unpublished) may also indicate the

presence of non-centric DNA. It is possible that all unit centromeres are not separated by non-centric DNA. The observation that SCEs within the centromere region can be detected using the CREST label has not been previously reported. Using the 5-bromodeoxyuridine/Giemsa stained (harlequin) chromosomes, Carrano and Wolff (1975) observed a direct relationship between the DNA content and the frequency of SCEs in the Indian muntjac chromosomes. They reported an unusually high frequency of exchanges at the junction of the arms and the long neck-like centromere region. However, this frequency was far lower within the neck-like region. In view of the fact that the centromere region is very

compact, it is not clear if such exchanges occur within the non-centromeric heterochromatic segments.

The segregation of the multicentric chromosomes is of interest in mutagenesis. A dicentric or multicentric chromosome may cause anaphase bridges/frag- ments or may segregate in an orderly manner. Wan- da11 (1994) has shown that two adjacently located centromeres with fully developed and functional kinetochores may act as one unit because of their attachment to the microtubules facing the same pole. This apparently is also true of a hexacentric chromosome in a rat cell line (Paweletz et al., 1989) as well as the present situation. In the muntjac, there is evidence that the microtubules attach to all three kinetochores of the X-chromosome and face the spindle poles in a manner as if the chromosome is monocentric (Brinkley et al., 1984). There is, however, no estimate of the maximum distance between the two functional centromeres which would permit the chromosome to segregate normally. It is also not known if a break between two centromeres of a multicentric chromosome would result in more than one stable chromosomes thus changing the basic number in a cell or a species. Indian muntjac, however, may provide the first evidence that multicentric chromosomes with all functional centromeres can exist in nature.

Acknowledgements

Authors are thankful to Drs. Bill Morgan for the

cell line, Bill Brinkley for the SH serum, Bill Earn- shaw for antiCENP-B, -C and Bob Margolis for the anti CENP-A antibodies.

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The centromeres of the Indian muntjac: Evidence for the existence of multiple centromeres?