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Human Molecular Genetics, 2003, Vol. 12, No. 23 3109-3121
DOI: 10.1093/hmg/ddg330
© 2003 Oxford University Press

Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns

Jeffrey M. Craig, Elizabeth Earle, Paul Canham, Lee H. Wong, Melissa Anderson and K.H. Andy Choo*

Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Melbourne, Victoria 3052, Australia

Received June 26, 2003; Revised September 15, 2003; Accepted September 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have examined the metaphase chromosomal localization of 15 proteins that have previously been described as involved in mammalian chromatin modification and/or transcriptional modulation. Immunofluorescence data indicate that all the proteins localize to human and mouse centromeres, a neocentromere, and the active centromere of a dicentric chromosome, with six of these proteins (Sin3A, PCAF, MYST, MBD2, ORC2, P300/CBP) being demonstrated at mammalian centromeres for the first time. Most of these proteins fall into two distinct chromosomal distribution patterns: (a) kinetochore-associated proteins (Sin3A, PCAF, MYST and BAF180), which colocalize with metaphase kinetochores, but not any of the pericentric and other major heterochromatic regions; and (b) heterochromatin-associated proteins (MeCP2, MBD1, MBD2, ATRX, HP1{alpha}, HDAC1, HDAC2, DNMT1 and DNMT3b), which colocalize with centromeric/pericentric heterochromatin and all other major heterochromatic sites. A heterogeneous third group (c) consists of the origin recognition complex subunit ORC2 and the histone acetyltransferase P300/CBP, which associate generally with kinetochores in humans and centromeric/pericentric heterochromatin in mouse, with some minor differences in localization. These observations indicate an extensive sharing of protein components involved in chromatin modification at gene loci, centromeres and various chromosomal heterochromatic landmarks. The definition of distinct patterns of chromosomal distribution for these proteins provides a useful basis for the further investigation of the broad-ranging roles of these proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Centromeres are responsible for the attachment of chromosomes to the mitotic and meiotic spindle, and are essential for chromosome segregation. They perform most of these functions via the kinetochore, which contains a variety of proteins with roles ranging from establishing a centromeric nucleosome structure to checkpoint control (1). Neocentromeres are fully functional centromeres that occur at ectopic genomic locations (2). Human neocentromeres contain no detectable {alpha}-satellite DNA that resides at typical human centromeres, and have been shown to bind all essential centromere proteins (3). Of note, despite the lack of long tracts of tandemly repeated DNA, human neocentromeres are heterochromatic in nature, as shown by their association with known heterochromatin proteins (3,4). These properties make neocentromeres a useful system to facilitate the study of centromere structure and function (5,6).

Most eukaryotic centromeres, including those of fission yeast, Drosophila and mammals, contain regions of heterochromatin; that is, chromatin packaged in a condensed state throughout the cell cycle (7). On a cytological scale, this heterochromatin may appear to overlap with the kinetochore (8–10), and in some organisms flank the kinetochore along the chromosome axis (11,12). Pericentric heterochromatin contains few transcribed genes and can form domains that are involved in gene silencing in trans within the interphase nucleus (13,14).

The traditional view of heterochromatin has been developed principally around Drosophila and mammalian pericentric heterochromatin that usually contain large tracts of tandem repeats (7). Human centromeres also contain pericentric heterochromatin, with several chromosomes exhibiting larger heterochromatic bands recognizable at the cytological level, at 1q12, 9q12, 16q11.2, Yq12 and the p-arms of all the acrocentric chromosomes. More recently, heterochromatin proteins have been found at silenced gene promoters (15) and human neocentromeres (3,4), indicating the need to broaden the definition of the heterochromatic status of a genomic region to reflect the presence of heterochromatin proteins (16).

Investigation of the protein components of the silent mating type loci and centromeres of fission yeast, and gene promoters and pericentric heterochromatin of higher eukaryotes, have led to the ‘histone code hypothesis’, whereby dynamic changes in the covalent modification of histones, such as acetylation and methylation, provide a code for the correct regulation of open (transcriptionally competent) or closed (transcriptionally silent) chromatin, mostly by affecting chromatin structure and interactions of non-histone proteins with chromatin (17). The two main types of epigenetic modifier proteins are those involved in the covalent modification of histones, such as histone deacetylases (HDACs) and histone acetyltransferases (HATs), and ATP-dependent chromatin remodelling factors that enable histone-DNA shuffling (7,18).

HDACs are central to establishing and maintaining silent chromatin. CpG-methylation ( meCpG) is another integral component of the silent chromatin. DNA methyltransferases (DNMTs), which maintain meCpG, and meCpG-binding proteins (MeCPs), both act as corepressors by interacting with HDACs (18,19). DNA-binding transcription factors such as those of the Ikaros family, can recruit HDACs directly to promoters or via corepressors such as CtBP or Sin3A (18,20). These factors form macromolecular complexes that cooperate with ATP-dependent chromatin remodelling factors to modify chromatin (18).

Some of the proteins involved with chromatin modification at gene promoters have been localized cytologically to centromeric regions (reviewed in 21). However, most studies have concentrated on interphase chromosomes, and many of these proteins remain untested for their interaction with the centromere. In this study, we have used antibodies to 15 different proteins, most of which are known to play major roles in the epigenetic control of gene expression, to determine their relationship with human and mouse kinetochores and pericentric heterochromatin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antibodies to 15 different proteins were used in immunofluorescence study. These proteins included the histone deacetylases (HDAC1 and HDAC2), DNA methyltransferases (DNMT1 and DNMT3b), MeCPs (MeCP2, MBD1 and MBD2), the transcriptional corepressor Sin3A, chromatin remodellers (ATRX and BAF180), origin recognition complex subunit ORC2, chromodomain protein HP1{alpha}, and histone acetyltransferases (HATs—PCAF, MYST and P300/CBP). All of these proteins, except methyl binding domain protein 1 (MBD1) and the chromatin remodellers, have been shown to be subunits of, or interact directly with, macromolecular chromatin protein complexes, and all proteins except the chromatin remodellers have previously been found at gene promoters (18,22,23).

It is noteworthy that, with a few exceptions, human chromosomes in general exhibit less heterochromatin than typical mouse chromosomes (24,25). The exceptions are the large bands of heterochromatin seen in the pericentric q-arm regions of human chromosomes 1, 9 and 16, the q12 region of Y, and the pericentric p-arm regions of the acrocentric chromosomes. For the purpose of illustration for our immunofluorescence results, we have used randomly selected mouse chromosomes, but for humans, we chose to use chromosome 1 (and acrocentric chromosomes in some cases) for consistency of displaying the protein distribution patterns for both the kinetochore and pericentric heterochromatin on the same chromosome. In addition, we also showed partial metaphase spreads containing examples of at least 10 random human or mouse chromosomes stained with anti-centromere antiserum (ACA) CREST3 or anti-CENP-A antibody. The results are summarized in Table 1 and described below.


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  		Table 1. Localization of proteins on normal metaphase, dicentric and neocentric chromosomes
 
A number of different mouse and human cell lines were used for immunofluorescence analysis (see Materials and Methods). These included a human cell line containing a dicentric chromosome with two well-separated repetitive centromeric heterochromatic regions, only one of which forms an active, kinetochore-bearing centromere (26). We also examined the localization of these antibodies on our well-characterized mardel (10) neocentromere (5,27–29). Antibodies were co-detected with antibodies against previously cytologically mapped centromere proteins: inner-kinetochore protein CENP-A (30) or ACA CREST3 (see Materials and Methods). In addition, we co-detected proteins with antibodies to outer-kinetochore mitotic checkpoint protein BubR1 (31), and heterochromatin proteins HP1{alpha} (32) or MBD1 (33). Analysis was initially based on cytological observations, which enabled us to categorize proteins as kinetochore- or heterochromatin-specific. We further characterized protein-distribution patterns in two ways. The first was to apply fluorescence intensity line scans (graphs of pixel intensity versus distance) laterally through sister kinetochores and longitudinally through the centromere along the chromosome axis (see below). In the second method, applicable only to kinetochore-associated proteins where distinct doublet kinetochore-signals were discernible [see Table 1 subgroups (a) and (c); and described below], we directly measured the inter-kinetochore distance for each signal-doublet of at least 40 chromosomes, and compared this distance with that of the co-detected CENP-A/CREST3 or the outer-kinetochore protein BubR1 (see Materials and Methods). This allowed us to determine whether a protein was localised significantly further apart than CENP-A (i.e. in an outer-kinetochore location), and to confirm such a conclusion using BubR1 colocalization measurements.

Kinetochore-associated proteins
Antibodies to Sin3A, PCAF, MYST and BAF180 all showed preferential staining at kinetochores on mouse and human metaphase spreads, but not to any significant extent at the pericentric or other heterochromatin (Table 1). Figures 1 and 2 show typical examples of immunofluorescence staining from two members of this group, PCAF and Sin3A, respectively.



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  		Figure 1. Immunofluorescence results showing distribution of PCAF on human or mouse chromosomes. For human chromosomes, examples of chromosome 1 (A, B, E) and a partial metaphase spread (I) are shown for the colocalization with known centromere proteins. For mouse, a randomly chosen chromosome (C, D, F) and a partial metaphase spread (J) are shown. Known centromere proteins were represented by ACA CREST3 (equivalent to inner-centromere protein CENP-A); the outer-kinetochore protein BubR1; heterochromatin protein MBD1 (or HP1{alpha} for some other proteins); and CREST6, an autoimmune serum containing anti-CENP-A and anti-CENP-B antibodies, and localizing to both the active (a) and inactive (i) centromeres of a dicentric chromosome. FISH indicates the signal from a mardel(10) neocentromere-specific BAC probe. Fluorescence intensity line-scans taken laterally (three pixels wide) and axially (13 pixels wide) through the kinetochore (as shown by arrows in K and L, respectively), are also presented. For each chromosome, multi-colour and split-colour images are shown: blue (DAPI) for the chromosome; red (Texas Red) for PCAF; and green (FITC) for known centromere proteins or FISH signal. PCAF is shown to localize on the lateral outside edges of CENP-A (A, C, I) and fully with BubR1 (B, D); to identify only a subset of pericentric heterochromatin (E, F); to localize to the mardel(10) neocentromere (G), and to the active centromere only of a human dicentric chromosome (H).

 


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  		Figure 2. Immunofluorescence results showing distribution of Sin3A on human or mouse chromosomes. Detailed explanations are as for Figure 1.

 
Antibodies to the kinetochore-associated proteins all localized at the lateral outside edges of the CREST3 signals on human centromeres (Figs 1A and I, 2A and I, and 3). Localization to the outer kinetochores was confirmed by colocalization with a known outer-kinetochore mitotic checkpoint protein BubR1 (31) (Figs 1B, 2B and 3). On mouse chromosomes, colocalization of these proteins to the outer kinetochore regions is more difficult to discern, due presumably to the tighter centromeric chromatin structures of these chromosomes (Figs 1 and 2C, D, and J). Using antibodies to heterochromatin proteins HP1{alpha} or MBD1, Sin3A, PCAF, MYST and BAF180 all consistently occupied a significantly smaller region especially axially, than HP1{alpha} or MBD1, at the mouse and human pericentric heterochromatin (Figs 1 and 2E and F).



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  		Figure 3. Measurement of inter-signal distances at human sister kinetochores. Distances between centres of fluorescent signals for all proteins localizing to human kinetochores were measured for at least 40 kinetochores, averaged, and compared to co-detected inner kinetochore CENP-A/ACA CREST3 or outer kinetochore BubR1. Distances were then then converted to distances relative to CENP-A or BubR1. Error bars of ±1 SD and P-values for each antibody/CENP-A pair are also shown on each graph. Sin3A, PCAF, MYST, BAF180 and P300/CBP were all found to localize significantly outer to CENP-A/CREST3 and to colocalize with BubR1, whereas ORC2 co-localized with CENP-A/CREST3 and was significantly inner to BubR1.

 
Immunofluorescence analysis of the mardel(10) chromosome showed positive signals at the neocentromere for each of the antibodies to this subgroup of proteins (Figs 1G and 2G; Table 1). Testing of all members of this group on the human dicentric chromosome further demonstrated binding only to the active centromere (Figs 1H and 2H; Table 1), except for MYST, where the determination of whether this protein was present or absent at the inactive centromere was prevented by relatively high background signals on the chromosome arms.

Heterochromatin-associated proteins
Antibodies to this subgroup of proteins, consisting of MeCP2, MBD1, MBD2, ATRX, HP1{alpha}, HDAC1, HDAC2, DNMT1 and DNMT3b, all exhibited a similar staining pattern in the different cell types investigated, as exemplified by DNMT1 and MBD2 in Figures 4 and 5, respectively. The results indicated that, unlike the discreet doublet signals of a kinetochore-associated protein, immunofluorescence staining for these proteins generally mirrored that of other known heterochromatin proteins, such as HP1{alpha} and MBD1, showing strong signals at both the centromeres and the large bands of pericentric heterochromatin in human and mouse chromosomes, including the short-arm heterochromatin of the human acrocentric chromosomes (Figs 4 and 5A–E), as described for MBD1, HP1{alpha} and HP1ß and ATRX (9,10,33,34). In addition, MBD2, HDAC1, HDAC2, DNMT1 and DNMT3b showed significant staining over whole chromosome arms, as was described for MBD1 (33).



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  		Figure 4. Immunofluorescence results showing distribution of DNMT1 on human and mouse chromosomes. For human chromosomes, examples of chromosome 1, an acrocentric chromosome, and a partial metaphase spread, are shown for colocalization with known centromere proteins (A, C, D, H); for mouse, a randomly chosen chromosome or a partial metaphase spread is shown (B, E, I). Known centromere proteins and FISH probe, and fluorescence intensity line-scans, are as detailed in Figure 1 except that anti CENP-A was used instead of CREST3. For each chromosome, multi-colour and split-colour images are shown: blue (DAPI) for the chromosome; red (Texas Red) for DNMT1; and green (FITC) for known centromere proteins or FISH signal. DNMT1 is shown to localize to a larger region than CENP-A (A, B, H, I); to colocalize with pericentric heterochromatin at human chromosome 1 (C), human acrocentric chromosomes (D), and mouse chromosomes (E); to localize to the mardel(10) neocentromere (F), and to both the active (a) and inactive (i) centromeres of a human dicentric chromosome (G).

 


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  		Figure 5. Immunofluorescence results showing distribution of MBD2 on human and mouse chromosomes. Detailed explanations are as for Figure 4.

 
When the binding properties of this subgroup of proteins were examined on the mardel(10) chromosome, an elevated doublet signal was observed at the neocentromere. The intensity of the neocentromere signal was consistently lower than at all other centromeres, but higher than that observed at the 10q25 region on the normal human chromosome 10 (Figs 4F and 5F; Table 1, and data not shown). We further determined the binding pattern of these proteins on the human dicentric chromosome, and showed a strong localization at both the active and inactive centromeres (Figs 4G and 5G); exceptions were the antibodies to HDAC1 and HDAC2, where high chromosome-arm background signals have prevented a clear visualization of these proteins at the inactive centromere (Table 1).

Kinetochore- and heterochromatin-associated proteins
In human cells, ORC2 and P300/CBP both localized to the kinetochores, but not to the pericentric heterochromatin (Figs 6A, F and J, and 7A, E and I). ORC2, however, was frequently observed at the satellites at the ends of the sort arm of the acrocentric chromosomes (Fig. 6B). In addition, when ORC2 was analysed against CENP-A and the outer-kinetochore protein BubR1, the protein was seen to colocalize with CENP-A at the inner kinetochores (Figs 6A and 3). In contrast, a similar analysis for P300/CBP against CREST3 and BubR1 in human cells indicated that this protein colocalized more with BubR1 (Figs 7A, B and 3).



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  		Figure 6. Immunofluorescence results showing chromosomal distribution of ORC2. For human chromosomes, examples of chromosome 1, an acrocentric chromosome, and a partial metaphase spread are shown (A, B, C, F, J); for mouse, a randomly chosen chromosome and a partial metaphase spread are shown (D, E, G, K). In human chromosomes, ORC2 is shown to colocalize with CENP-A (A, B, J) and slightly on the lateral inside edges of BubR1 (C), and identified a smaller region than heterochromatin protein MBD1 (F) and frequently to human acrocentric short arm satellites (B). In mouse chromosomes, however, ORC2 identified a larger region than CENP-A and BubR1 (D, E, K) and colocalized with pericentric heterochromatin protein MBD1 (G). ORC2 also localized to the mardel(10) neocentromere (H) and to the active (a) and inactive (i) centromeres of a human dicentric chromosome (I).

 


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  		Figure 7. Immunofluorescence results showing distribution of P300/CBP on representative human and mouse chromosomes. Detailed explanations are as for Figure 6, except that P300/CBP did not normally localize to human acrocentric short arm satellites (data not shown) or to the inactive centromere (i) of the dicentric chromosome (H).

 
The immunofluorescence distribution patterns on mouse chromosomes was considerably different to those seen on the human chromosomes, where ORC2 and P300/CBP binding was detected at both the centromere and throughout the pericentric heterochromatin, reflecting both centromere and heterochromatin association (Figs 6D, E, G and K and Figs 7C, D, F and J). Immunofluorescence analysis also showed that both the proteins were enhanced at the mardel(10) neocentromere (Figs 6H and 7G) and the active centromere of a human dicentric chromosome (Fig. 6I and 7H). However, ORC2 was also present at the inactive centromere of the dicentric chromosome (Fig. 6I).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Recent studies have identified an increasing number of proteins responsible for the epigenetic modification of chromatin, in particular in establishing and maintaining active or silent chromatin found at gene promoters (reviewed in 7,17,18). The possible involvements of these proteins at the centromere and cytologically defined heterochromatin have been less well studied. In order to shed light on such possible involvements, we have investigated the detailed distribution of 15 of these proteins on human and mouse metaphase chromosomes. These proteins include those involved in covalent modification of histones (HDACs and HATs), chromatin remodelling (BAF180 and ATRX), meCpG-maintenance or binding (DNMTs, MeCPs), transcriptional corepression (Sin3A), heterochromatin formation (Hp1{alpha}) and origin binding (ORC2). Our results have identified all 15 proteins to be enriched at the human and mouse centromeres, with most of these proteins falling into two general chromosomal distribution subgroups (Table 1).

The first chromosomal subgroup, consisting of Sin3A, PCAF, MYST and BAF180, localize to active kinetochores only. All members bind to outer kinetochores except BAF180, which colocalizes with CENP-A. Apart from BAF180, localization of the other three members to mammalian centromeres has not previously been described. The similar chromosomal distribution pattern of the histone H3 methyltransferase SUV39H1 (35) suggests that this protein also belongs to this subgroup. These results therefore define a substantial subgroup of chromatin modifier proteins whose binding is linked to functional kinetochores.

The second subgroup of proteins, comprising MeCP2, MBD1, MBD2, ATRX, HP1{alpha}, HDAC1, HDAC2, DNMT1 and DNMT3b localize, at the current level of resolution, to centromeres as well as all major mouse and human regions of pericentric and non-pericentric heterochromatin, and the inactive dicentric centromere. These results confirm previous localization of MeCP2, MBD1, ATRX and HP1{alpha} on the pericentric heterochromatin of mammalian metaphase chromosomes (10,33,34,36), and extend the previously observed interphase localization of HDAC1, HDAC2, DNMT1, and DNMT3b (14,37–39) to metaphase chromosomes. In addition, the results reveal for the first time the localization of MBD2 at mammalian pericentric and kinetochore regions.

The apparently indiscriminate binding of this subgroup of proteins to all major sites of cytological heterochromatin is in stark contrast to the behaviour of proteins of subgroup (a). These heterochromatic sites are known to contain large arrays of a variety of tandem repeats, such as centromeric mouse minor and pericentric major satellites, and human {alpha}- and ß-satellites and classical satellites I–III (25). This apparent sequence-independent mode of chromosomal association may be related to the unique tertiary structures presented by tandemly repeated DNA arrays, and/or chromatin-related macromolecular complexing of these proteins at these sites.

Localization of ORC2 and P300/CBP to mammalian centromeres has not been previously described. These proteins appear to exhibit some of the kinetochore-binding properties of subgroup (a), especially with human chromosomes, and some of the heterochromatin-binding properties of subgroup (b), especially mouse chromosomes.

What are the roles of these proteins at the centromere, and the significance of the different patterns of chromosomal distribution? The detection of all proteins at the mardel(10) neocentromere clearly indicates that the presence of large arrays of heterochromatic satellite DNA is not a prerequisite for the binding of any of these proteins. Indeed, this finding establishes a direct functional link between the establishment of centromere activity and the recruitment and assembly of these proteins. Since the roles of these proteins have previously been studied mostly in relation to transcriptional regulation, their specific roles at the centromere are largely unclear at present. It has been suggested that one of the members of this subgroup, SUV39H1, in addition to performing a role in setting up silent chromatin at centromeres, could also be involved in centromere-directed alignments of chromosomes as they move to the metaphase plate and/or play a role in regulating higher-order structure of the kinetochore (4). It is therefore possible that the other proteins in subgroup (a) may perform similar or related functions.

Amongst the six new mammalian centromere-binding proteins we have described, we have identified a functionally related class that has not previously been localized to centromeres. These are the histone acetyltransferases PCAF, MYST and P300/CBP. Why would proteins usually involved in forming active chromatin be present at centromeres? One explanation is that acetylation levels are the result of a dynamic equilibrium between HDACs and HATs and therefore both groups of proteins are expected to show overlapping localization (40). In addition, it has been shown that PCAF and HDAC1 interact in vivo and that HATs are integrated into a large multiprotein HDAC complex (41).

The loss of DNMT3b has been shown to result in ICF syndrome (42), which manifests at the cytological level as decondensation and instability of the major regions of human pericentric heterochromatin (reviewed in 43). The loss of heterochromatin proteins has also been shown to result in a number of other genetic diseases: e.g. Rett syndrome due to MeCP2 (44), ATRX syndrome due to ATRX (34), and Townes–Brocks syndrome due to SALL1 (45). The possibility that disruption in pericentric heterochromatin organization may play a significant part in the aetiology of these diseases should be a useful area of further investigation.

In summary, we have demonstrated an association with mammalian metaphase centromeres of 15 proteins previously reported to participate in a wide range of roles in chromatin and transcriptional modulation. Of these proteins, six have not previously been described at mammalian centromeres, while the remaining ones have been reported as present on either metaphase or interphase centromeres (10,21,33,34,36,46). Other chromatin-associated proteins that have been reported to bind mammalian pericentric heterochromatin, and which are not included in the present study, include the HP1 isoform HP1ß (9,10); its binding partner SUV39H1 (35), and chromatin target, MeH3K9 (47); the histone variant H2A.Z (48); transcriptional corepressors TIF1ß, KRAZ1, KRAZ2, SALL1; and the chromatin remodellers ACF1 and WSTF (reviewed in 21). Together, these observations point to the extensive sharing of protein components, many likely to be acting cooperatively through macromolecular complexes, at gene loci, centromeres and various chromosomal heterochromatic landmarks. The classification of these protein components into three distinct chromosomal distribution subgroups should provide a useful basis for the further delineation of the broad-ranging and largely unknown roles of these proteins at the different chromosomal locations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell lines
We used a lymphoblastoid cell line established from a patient with the mardel(10) neocentromere (27), maintained in RPMI 1640 medium (Trace Biosciences, Australia) supplemented with 20% fetal calf serum (FCS); a somatic cell hybrid [ES-mardel(10)-1] made by transferring the mardel(10) chromosome into the mouse ES cell line ES129-1, maintained in ES cell medium (Trace) supplemented with ß-mercaptoethanol and LIF; a dicentric lymphoblastoid cell line from a patient with a t(X;15) translocation (26) maintained in RPMI 1640 medium (Trace) supplemented with 20% FCS; and a transformed mouse T cell line VL3-3M2, maintained in RPMI 1640 medium (Trace) supplemented with 5% FCS. Cells were arrested in metaphase using standard Colcemid treatment.

Antibodies and immunofluorescence
The specificity of all antibodies used in this study had been previous tested by one or more of the following techniques: western blotting, immunoprecipitation, immunohistochemistry and ELISA. Human anti-centromere antisera (ACA) were provided and characterized by S. Wittingham and T. Kaye, Royal Melbourne Hospital. ACA CREST3 recognized primarily CENP-A, and ACA CREST6 recognized primarily CENP-A and CENP-B (27). Rabbit anti-mouse CENP-A, and rabbit anti-human CENP-A antibodies have been described elsewhere (27,49,50). CREST3 was used in place of anti CENP-A when co-detected with antibodies raised in rabbits and for consistency when comparing inner or outer centromere localization; CREST6 was used to identify both centromeres of dicentric chromosomes. Other antibodies used were as follows (with dilutions and preferred buffers, see below): mouse anti human ATRX (23c or 39f), from Doug Higgs, MRC Molecular Haematology Unit, IMM, Oxford, UK [1 : 20, conditions as McDowell et al. (34)]; rabbit anti human BAF180 from Weidong Wang, Laboratory of Genetics, NIH, Baltimore, MD, USA (1 : 100, KCM); goat anti human DNMT1, from Santa Cruz (1 : 25, TEEN+) or mouse anti human DNMT1 from Imgenex, San Diego, CA, USA (1 : 25, TEEN+); goat anti human DNMT3b, from Santa Cruz (1 : 25, TEEN+) or mouse anti mouse DNMT3b, from Imgenex (25, TEEN+); rabbit anti human HDAC1, from Calbiochem, San Diego, CA, USA (1 : 50, TEEN+) or goat anti human HDAC1, from Santa Cruz (1 : 100; TEEN+); rabbit anti human HDAC2, from Calbiochem (1 : 50, TEEN+) or goat anti human HDAC2, from Santa Cruz (1 : 100; TEEN+); mouse anti human HP1{alpha} (clone 2HP1H5), from Pierre Chambon, CNRS, INSERM, Strasbourg, France [1 : 250; conditions as Minc et al. (54)]; sheep anti human MBD1, from Brian Hendrich, ICMB, University of Edinburgh, UK (1 : 200, TEEN+); sheep anti human MBD2, from Brian Hendrich (1 : 100, TEEN+) or mouse anti human MBD2 from Imgenex (1 : 50, TEEN+); rabbit anti rat MeCP2, from Brian Hendrich (1 : 200, TEEN+) or rabbit anti mouse MeCP2, from Upstate Biotechnology, Waltham, MA, USA (1 : 200, TEEN+); rabbit anti yeast MYST family, from Upstate Biotechnology (1 : 100, TEEN+); mouse anti human ORC2 (Ab-2), from Calbiochem (1 : 20, TEEN) or mouse anti human ORC2, from NeoMarkers, Fremont, CA, USA (1 : 50, TEEN+); mouse anti human P300/CBP (mixed epitope), from Upstate (1 : 25, TEEN+) or mouse anti human P300/CBP (RW128), from Upstate (1 : 50, TEEN+); mouse anti human PCAF, from Santa Cruz (1 : 25, KCM) or rabbit anti human PCAF, from Upstate (1 : 25, TEEN+); rabbit anti mouse mSin3A (AK-11), from Santa Cruz (1 : 100, TEEN+) or rabbit anti mouse mSin3A, from Upstate (1 : 25, TEEN+). Table 2 summarizes the quality control information (i.e. whether affinity purified, and/or analysed by western blotting, immunohistochemistry or ELISA) for each of the proteins that have not previously been observed at metaphase centromeres (see Results and Table 1), and indicates that, with the exception of MYST, our results were confirmed with antibodies from two different sources.


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  		Table 2. Quality control data for proteins not previously localized to mammalian metaphase centromeres (see Table 1)
 
Immunofluorescence was carried out on unfixed, permeablized cells according to published methods (26,51,52) with minor modifications. Antibodies were diluted either in TEEN+ [1 mM triethanolamine–HCl (pH 8.5), 0.2 mM NaEDTA (pH 9.0), 25 mM NaCl, 0.1% Triton X-100, 0.1% BSA] or KCM [20 mM KCl; 20 mM NaCl; 10 mM Tris–HCl; 0.5 mM NaEDTA; 0.1% (v/v) Triton X-100]. An exception was the goat primaries, where 5% donkey serum was substituted for BSA in the TEEN+. All washes were performed using KCM and antibodies were detected using fluorescently labelled secondaries (Jackson Immunoresearch Laboratories, West Grove, PA, USA) and in the case of ATRX, HP1 (on human chromosomes) and ORC2, a fluorescently labelled tertiary antibody following an unlabelled secondary. Antibodies were detected with Texas Red-conjugated secondaries and were co-localized with FITC-detected ACA CREST3 or anti-CENP-A, or ACA CREST6 for dicentric chromosomes and antibodies against HP1{alpha} (or MBD1 for primaries raised in mouse) to stain pericentric heterochromatin. Immuno-FISH was carried out using the mardel(10) neocentromere-specific BAC E8 as previously described (27) and detected using FITC-labelled antibodies. Images were captured using IPLab software (Scanalytics, Fairfax, VA, USA). Graphs of total pixel intensity versus distance (line scans) were generated using the IPLab extension Line Measure with a pixel width of 3 for lateral scans across kinetochores and 13 for axial scans through centromeres (as illustrated in Fig. 1K and L). The distances between kinetochore doublet-signals for all the chromosomes within a single metaphase spread were measured using IPLab and Adobe Photoshop. Student's t-test was applied to co-localization data-sets and a p-value of 0.05 was used as a cut-off above which two proteins were concluded to coincide.


   ACKNOWLEDGEMENTS
 
We thank L. Shaffer for MES10 and dicentric cell lines, respectively; S. Smale for cell line VL3-3M2; P. Kalitsis for the CENP-A antibody; D. Higgs and T. McDowell for ATRX antibodies; W. Wang and D. Murray for BAF180 antibody; P. Chambon for HP1{alpha} antibody; and B. Hendrich for MBD1, MBD2 and MeCP2 antibodies. J.M.C. and K.H.A.C. are associates of the Department of Paediatrics, University of Melbourne. K.H.A.C. is a Senior Principal Research Fellow of NH&MRC of Australia. This work was supported by funding from NH&MRC.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +61 383416306; Fax: +61 393481391; Email: choo{at}cryptic.rch.unimelb.edu.au Back


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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