Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads

Abstract

We have screened for the presence of two centromere autoantigens, CENP-B (80 kDa) and CENP-C (140 kDa) at the inactive centromere of a naturally occurring stable dicentric chromosome using specific antibodies that do not cross-react with any other chromosomal proteins. In order to discriminate between the active and inactive centromeres on this chromosome we have developed a modification of the standard methanol/acetic acid fixation procedure that allows us to obtain high-quality cytological spreads that retain antigenicity with the anti-centromere antibodies. We have noted three differences in the immunostaining patterns with specific anti-CENP-B and CENP-C antibodies. (1) The amount of detectable CENP-B varies from chromosome to chromosome. The amount of CENP-C appears to be more or less the same on all chromosomes. (2) CENP-B is present at both active and inactive centromeres of stable dicentric autosomes. CENP-C is not detectable at the inactive centromeres. (3) While immunofluorescence with anti-CENP-C antibodies typically gives two discrete spots, staining with anti-CENP-B often appears as a single bright bar connecting both sister centromeres. This suggests that while CENP-C may be confined to the outer centromere in the kinetochore region, CENP-B may be distributed throughout the entire centromere. Our data suggest that CENP-C is likely to be a component of some invariant chromosomal substructure, such as the kinetochore. CENP-B may be involved in some other aspect of centromere function, such as chromosome movement or DNA packaging.

Human centromere repositioning “in progress”

“…CENP-B, on the other hand, was retained at the 4p10 ␣-satellite and was not evident at the neocentromere site (Figs. 2C and 5), consistent with the known specificity of CENP-B for CENP-Bbox-containing ␣-satellite irrespective of its centromere activity status (13). Heterochromatin protein HP1 was observed to bind to both the active and inactive centromeres (Fig.…”

CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase

“…Autoimmune serum GS, which was used for the initial identification of CENP-A, -B, and -C (Earnshaw and Rothfield, 1985), was affinity purified against protein encoded by k gtll clone CNPC3 (Saitoh et al, 1992) as described in Earushaw et al (1989). This clone produces a partial CENP-C peptide containing amino acid residues 296-943.…”

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

“…Independent of this sequence preference, specific deposition of the centromeric histone H3 variant CENP-A (Earnshaw and Rothfield, 1985) is thought to form the basis for an 'epigenetic' maintenance of centromere identity (Allshire and Karpen, 2008;Okamoto et al, 2007;Vafa and Sullivan, 1997;Warburton et al, 1997). The epigenetic control of centromere activity is strikingly illustrated by the inactivation of centromeres on dicentric chromosomes (Earnshaw and Migeon, 1985;Earnshaw et al, 1989;Merry et al, 1985;Sugata et al, 2000;Sullivan and Schwartz, 1995) and by rare neocentromeres that recruit CENP-A and assemble fully functional kinetochore structures on non-alphoid DNA (Alonso et al, 2007;Depinet et al, 1997;du Sart et al, 1997;Saffery et al, 2000;Warburton et al, 1997).…”