Journal of Cell Science 109, 2199-2206 (1996) 2199 Printed in Great Britain © The Company of Biologists Limited 1996 JCS3386 Epigenetic control of mammalian centromere protein binding: does DNA methylation have a role? Arthur R. Mitchell*, Peter Jeppesen, Linda Nicol†, Harris Morrison and David Kipling MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK *Author for correspondence (internet [email protected]) †Present address: MRC Reproductive Biology Unit, Edinburgh, UK SUMMARY Chromosome 1 of the inbred mouse strain DBA/2 has a block of minor satellite DNA sequences on chromosome 1. polymorphism associated with the minor satellite DNA at The binding of the CENP-E protein does not appear to be its centromere. The more terminal block of satellite DNA affected by demethylation of the minor satellite sequences. sequences on this chromosome acts as the centromere as We present a model to explain these observations. This shown by the binding of CREST ACA serum, anti-CENP- model may also indicate the mechanism by which the B and anti-CENP-E polyclonal sera. Demethylation of the CENP-B protein recognises specific sites within the arrays minor satellite DNA sequences accomplished by growing of minor satellite DNA on mouse chromosomes. cells in the presence of the drug 5-aza-2′-deoxycytidine results in a redistribution of the CENP-B protein. This protein now binds to an enlarged area on the more terminal Key words: Centromere satellite DNA, Demethylation, Centromere block and in addition it now binds to the more internal antibody INTRODUCTION A common feature of many mammalian pericentromeric domains is that they contain families of repetitive DNA The centromere of mammalian chromosomes is recognised at sequences (Singer, 1982). Some of these families of repeated the cytological level as the primary constriction of the chro- DNAs appear to be quite specific for any one species. In man, mosome. Using the electron microscope Rattner and Bazett- these are the simple-sequence, so-called satellite DNAs, where Jones (1989) and Ris and Wit (1981) identified the kinetochore particular chromosomes have amplified relatively short as an electron dense component of the centromere with specific oligonucleotides to give the present-day situation where they structural features. These included inner and outer plates and may contain millions of copies of these repetitive DNA a fibrous corona. Phosphorous was one of the elements families (Prosser et al., 1986). A similar situation can be seen detected in this structure indicating that DNA was one of the in the mouse. In Mus musculus both the major and minor structural components. Later, Cooke et al. (1993) came to the satellite DNAs are present on all chromosomes with the conclusion that both the outer plate of the kinetochore and the exception of the Y chromosome (Pardue and Gall, 1969; Jones, matrix between the outer and inner plates either lacked DNA 1970; Wong and Rattner, 1988; Joseph et al., 1989). The same as an integral part of their structure or, if DNA sequences were is true for Mus spretus although the amount of DNA homolo- present, only a small amount (~2 kb) of DNA was involved gous to the Mus musculus major satellite DNA sequences in with these structural components. The initial finding that phos- the M. spretus genome is greatly reduced (Narayanswami et phorous was present in the outer plate being ascribed more al., 1992). In Mus caroli different families of repetitive DNAs likely to the presence of phosphoproteins rather than DNA have evolved within the centromeric domains of its chromo- sequences (Cooke et al., 1993). somes, and are quite distinct from those in the other two Mus The biological role of the centromere in the cell is of species (Kipling et al., 1995). paramount importance to the chromosome. The loss of cen- It was known that certain families of repetitive DNAs tromere function leads to chromosome instability during cell mapped, by in situ hybridisation, closer to the primary con- division which in turn leads to chromosome loss. Kinetochores striction of chromosomes than others (Mitchell et al., 1985; are recognised as the attachment sites for the spindle micro- Wong and Rattner, 1988; Joseph et al., 1989). Little sequence tubules. The loss of centromere function is associated with the similarity was found between the alphoid DNA in man and the chromosome being unable to capture microtubules. Thus during minor satellite DNA in M. musculus. Masumoto et al. (1989) cell division the alignment onto the metaphase plate and correct were first to report that both of these repetitive DNAs contained segregation of the chromosome during anaphase is prevented a conserved 17 base pair (bp) motif and that this short sequence (Schulman and Bloom 1991; Rattner, 1991). Errors of this type was responsible for binding the CENP-B centromere protein. can lead to complex genetic disorders in higher organisms. CENP-B is one of the proteins detected by autoantibodies in the 2200 A. R. Mitchell and others serum of some patients with the complex syndrome termed is an important component of some mammalian centromeres CREST (Moroi et al., 1980). Comparison of the CENP-B then the prevention of its binding by DNA methylation may be protein from man and mouse (Sullivan and Glass, 1991) has sufficient to cause centromere inactivation. shown 95% conservation at the amino acid level and from an analysis of its structure Pluta et al. (1992) initially showed that MATERIALS AND METHODS a DNA binding domain is present at the amino terminus. The first 158 amino acids were necessary to target the protein to the Chromosome preparations for indirect centromere. Later, Kitagawa et al. (1995) confirmed these immunofluorescence microscopy results and went on to show that the CENP-B protein had a DBA/2 mice were maintained in Edinburgh. Chromosome prepara- protein-protein dimerisation domain at its carboxy terminus. tions from spleen were prepared as described previously (Mitchell et Because it contains these different functional domains it has al., 1993). Cells were accumulated in mitosis and metaphase spreads been proposed that its biological role within both the alphoid were prepared as described by Jeppesen et al. (1992). Demethylation arrays in man and the minor satellite arrays in M. musculus is of chromosomal DNA was carried out by culturing cells in the presence of 5-aza-2′-deoxycytidine (Sigma) for 72 hours. The to promote condensation of the chromatin containing these optimum condition for growing the lymphocytes in the presence of repetitive DNAs (Kitagawa et al., 1995). this drug was found to be 5×10−6 M. After a period of 36 hours the The relationship between CENP-B and the kinetochore cell cultures were replenished with one half volume of fresh medium remains unclear. The CENP-B protein appears localised to the containing 5-aza-2′-deoxycytidine at the above concentration. Control chromatin beneath the kinetochore plate (Pluta et al., 1990) and cultures were set up at the same time and grown under identical con- it does not seem to be directly associated with this structure. ditions. Both control and treated cells were prepared at the same time Where dicentric chromosomes have been studied it now seems and the antibody reactions were run in parallel. clear that CENP-B can be bound at both active and inactive Indirect immunofluorescence with antisera against CENP-B or centromeres (Earnshaw et al., 1989; Sullivan and Schwartz, CENP-E was carried out essentially as described previously (Jeppesen 1995). In contrast the CENP-C protein appears to be associ- et al., 1992). ated only with the active centromere/kinetochore as deter- Anti-CENP-B raised against cloned human CENP-B (a gift from Professor W. C. Earnshaw) and anti-human CENP-E (a gift from Dr mined by the position of the primary constriction on chromo- Tim Yen) were both polyclonal rabbit antisera raised against bacteri- somes (Earnshaw et al., 1989; Page et al., 1995; Sullivan and ally expressed protein. Schwartz, 1995). The CENP-E protein also seems to bind only Anti-CENP-B or anti-CENP-E were diluted 1:300 or 1:500, respec- to the active kinetochore (Sullivan and Schwartz, 1995). In our tively, in KCM (Jeppesen et al., 1992) + 10% normal goat serum previous work on the DBA/2 mouse (Mitchell et al., 1993) we (NGS). The secondary antibody was FITC-conjugated, affinity- showed that chromosome 1 in this animal differed from the purified, anti-rabbit IgG, raised in goat (Sigma Chemical Co.), diluted other chromosomes in the cell by having two separate blocks 1:20 in KCM + 10% NGS. For double antibody labelling, slides were of minor satellite DNA sequences. These blocks are cytologi- first incubated with KCM + 10% NGS containing a 1:100 dilution of cally distinct and separated from each other by intervening a CREST patient anti-centromere serum (CP) together with either major satellite DNA sequences. Only the more terminal of the anti-CENP-B or anti-CENP-E antiserum (both diluted 1:100). After two arrays has the characteristics of a functional centromere, washing, slides were incubated simultaneously with FITC-conju- gated, affinity-purified, anti-human IgG, raised in goat (Sigma i.e. CREST anti-centromere associated labelling (ACA) and Chemical Co.) to detect the CREST signal, and TRITC goat anti- sister chromatid attachment. However, in situ hybridisation rabbit IgG to detect rabbit antibody binding, both diluted 1:20 in KCM results revealed the presence of 17 bp CENP-B binding sites + 10% NGS. within both arrays of minor satellite DNA. The presence of CENP-B sites in the more internal array conflicted with the Primed in situ hybridisation (PRINS) apparent absence of CREST ACA staining to this region. The original technique of Koch et al.